The partial amino acid sequence of bovine cartilage proteoglycan, deduced from a cDNA clone, contains numerous Ser-Gly sequences arranged in homologous repeats.

We have determined the sequence of a partial cDNA clone encoding the C-terminal region of bovine cartilage aggregating proteoglycan core protein. The deduced amino acid sequence contains a cysteine-rich region which is homologous with chicken hepatic lectin. This lectin-homologous region has previously been identified in rat and chicken cartilage proteoglycan. The bovine sequence presented here is highly homologous with the rat and chicken amino acid sequences in this apparently globular region. A region containing clusters of Ser-Gly sequences is located N-terminal to the lectin homology domain. These Ser-Gly-rich segments are arranged in tandemly repeated, approx. 100-residue-long, homology domains. Each homology domain consists of an approx. 75-residue-long Ser-Gly-rich region separated by an approx. 25-residue-long segment lacking Ser-Gly dipeptides. These dipeptides are arranged in 10-residue-long segments in the 100-residue-long homology domains. The shorter homologous segments are tandemly repeated some six times in each 100-residue-long homology domain. Serine residues in these repeats are potential attachment sites for chondroitin sulphate chains.     

Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.     

Proteoglycan-Lb, a small dermatan sulfate proteoglycan expressed in embryonic chick epiphyseal cartilage, is structurally related to osteoinductive factor.

We have isolated cDNA clones encoding the core protein of PG-Lb, proteoglycan which has been shown to be preferentially expressed in the zone of flattened chondrocytes of the developing chick limb cartilage (Shinomura, T., Kimata, K., Oike, Y., Yano, S., and Suzuki, S. (1984) Dev. Biol. 103, 211-220). The deduced amino acid sequence from the cDNA analysis indicates the presence of consensus leucine-rich repeats which are present in other small proteoglycans, decorin, biglycan, and fibromodulin. However, the homology analysis revealed that chick PG-Lb showed a higher homology (about 50% in the region containing leucine-rich repeats) to human osteoinductive factor, OIF, rather than to the other small proteoglycans. Furthermore, 6 cysteine residues are detected in both PG-Lb and OIF with invariant relative positions. Therefore, such an evolutionarily conserved structure in the PG-Lb core protein might be involved in some important biological functions of this molecule. In close relation to the structural similarity to OIF, the unique expression of PG-Lb in the ossifying area of cartilage suggested the possible participation of this proteoglycan in osteogenic processes.     

Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone.

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.     

Molecular cloning of a multifunctional chicken protein acting as the prolyl 4-hydroxylase beta-subunit, protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. Comparison of cDNA-deduced amino acid sequences with those in other species.

Several recent studies indicate that a single polypeptide may act as the beta-subunit of prolyl 4-hydroxylase, the enzyme protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. We report here the isolation and characterization of cDNA clones encoding this multifunctional protein in the chicken. All the coding sequences were determined on the basis of nucleotide sequencing of five cDNA clones and amino acid sequencing of the N-terminal end of the chicken beta-subunit. The processed polypeptide contains 493 amino acid residues, the size of the respective mRNA being about 2.7 kb. The chicken beta-subunit cDNA sequences were 78% homologous to the previously reported human beta-subunit cDNA sequences at the nucleotide level and 85% homologous at the amino acid level. The homology of the chicken beta-subunit sequences to those reported for bovine thyroid-hormone-binding protein and rat protein disulphide-isomerase was also 85% at the amino acid level. Primary-structure comparisons between the four species indicated that the two proposed active sites of protein disulphide-isomerase, the two Trp-Cys-Gly-His-Cys-Lys sequences, are located within highly conserved regions, which are also homologous to the active sites of a number of thioredoxins. The middle of the polypeptide has an additional conserved region 100 amino acid residues in length in which the degree of homology between the four species is 94% at the amino acid level. This long conserved region may also be important for some of the multiple functions of the protein. The four extreme C-terminal amino acids of the polypeptide in all four species are Lys-Asp-Glu-Leu, a sequence that has been suggested to function as a signal for the retention of a protein in the endoplasmic reticulum.     

Cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for bovine preproinsulin

A cDNA library was prepared from poly(A+) RNA isolated from fetal bovine pancreas. Bacterial colonies were screened for sequences homologous to a rat preproinsulin I cDNA probe. Ten positive clones were selected at random and further studied. Northern blot analyses revealed that seven of these clones hybridized to a single RNA species, of approximately 400 nucleotides. Sequence analysis of one of these clones (pbI2885) revealed the entire structural region of bovine preproinsulin mRNA including a 72 nucleotide region encoding a signal peptide enriched in hydrophobic residues. The overall nucleotide homology between bovine and human preproinsulin mRNA was 76% for the preregion, 89% for the A chain, 83% for the B chain, and 68% for the C peptide (including a 15 nucleotide deletion).     

Molecular cloning of a new human G protein. Evidence for two Gi alpha-like protein families

The amino acid sequence of a novel G protein alpha subunit (Gx alpha) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL-60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (Mr 40,519) which is closely related to Gi alpha proteins. The amino acid sequence homology between Gx alpha and human myeloid Gi alpha is 86% with 15 nonconservative substitutions. Gx alpha also shares 86% homology with both rat brain and mouse macrophage Gi alpha but is more homologous (94%) to bovine brain Gi alpha with only 5 nonconservative amino acid differences. G proteins previously termed Gi alpha may fall into at least two distinct groups, with one including human myeloid Gi alpha, rat brain Gi alpha and mouse macrophage Gi alpha; and other Gx alpha and bovine brain Gi alpha. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined.     

Molecular cloning and the complete nucleotide sequence of cDNA to mRNA for S-100 protein of rat brain.

The complete nucleotide sequence of mRNA for beta-subunit of rat brain S-100 protein was determined from recombinant cDNA clones. The sequence was composed of 1488 bp which included the 276 bp of the complete coding region, the 120 bp of the 5'-noncoding region and the 1092 bp of the 3'-noncoding region containing two polyadenylation signals. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was homologous to the amino acid sequence of bovine S-100 beta subunit except 4 residues showing species differences. From the viewpoint of evolutionary implications, the homology between the nucleotide sequence of S-100 and those of rat intestinal Ca-binding protein (ICaBP) and calmodulin (CaM) was examined. A dot-blot hybridization of poly(A) RNA from the developing rat brains using a labeled cDNA showed a rapid increase in S-100 mRNA at 10-20 postnatal days. The presence of S-100 mRNA in C-6 glioma cells is also described.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species

The small proteoglycans (PG) of bone consist of two different molecular species: one containing one chondroitin sulfate chain (PG II) and the other, two chains (PG I). These two proteoglycans are found in many connective tissues and have Mr = 45,000 core proteins with clear differences in their NH2-terminal sequences. Using antisera produced against synthetic peptides derived from the human PG I and PG II NH2 termini, we have isolated several cDNA clones from a lambda gt11 expression library made against mRNA isolated from human bone-derived cells. The clones, which reacted with antisera to the PG II peptide, were sequenced and found to be identical with the PG II class of proteoglycan from human fibroblasts known as PG-40 or decorin. The clones reacting to the PG I antisera, however, had a unique sequence. The derived protein sequence of PG I showed sufficient homology with the PG II sequence (55% of the amino acids are identical, with most others involving chemically similar amino acid substitutions) to strongly suggest that the two proteins were the result of a gene duplication. PG II (decorin) contains one attached glycosaminoglycan chain, while PG I probably contains two chains. For this reason, we suggest that PG I be called biglycan. The biglycan protein sequence contains 368 residues (Mr = 42,510 for the complete sequence and Mr = 37,983 for the secreted form) that appears to consist predominantly of a series of 12 tandem repeats of 24 residues. The repeats are recognized by their conserved leucines (and leucine-like amino acids) in positions previously reported for a diverse collection of proteins (none of which is thought to be proteoglycans) including: two morphogenic proteins (toll and chaoptin) in the fruit fly; a yeast adenylate cyclase; and two human proteins, the von Willebrand Factor-binding platelet membrane protein, GPIb, and a rare serum protein, leucine-rich glycoprotein.     

Immunological identification of rat tissue kallikrein cDNA and characterization of the kallikrein gene family

A tissue kallikrein cDNA was identified by direct immunological screening with affinity-purified anti-rat tissue kallikrein antibody from a rat submandibular cDNA library constructed with the expression vector pUC8. Sequence analysis of the kallikrein cDNA revealed an encoded protein 97% homologous to the partial amino acid sequence of rat submandibular kallikrein. This cDNA was used to hybrid-select kallikrein-specific RNA from submandibular gland. Translation of the hybrid-selected RNA in a cell-free assay system resulted in the production of a 37 kDa peptide representing the preproenzyme. In addition, hybrid-selection of RNA under less stringent conditions showed cross-hybridization with other submandibular gland mRNA species. In correlation with these results, analysis of rat genomic DNA showed extensive hybridization, suggesting a family of closely related kallikrein-like genes. Consequently, a Charon 4A rat genomic library was screened for kallikrein genes by hybridization with rat tissue kallikrein cDNA. Thirty-four clones were isolated and found to be highly homologous by hybridization and restriction enzymes analyses. Fourteen unique clones were identified by restriction enzyme site polymorphisms within DNA segments which hybridized to the kallikrein cDNA probe and it was estimated that at least 17 different kallikrein-like genes are present in the rat. Sequence and structural analysis of one of the genomic clones revealed a gene structure similar to that of other serine proteinases. Comparison of the partially sequenced exon regions of the gene with the sequence of rat tissue kallikrein cDNA reveals 89% identity when aligned for the greatest homology. However, the genomic sequence predicts termination codons in all three translational reading frames, implying that this gene is nonfunctional, i.e., a pseudogene. Comparison of the rat genomic sequence to a kallikrein-like gene from the mouse reveals extensive preservation of exons, less identity within introns and no significant homology between extragenic regions.