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Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.

Type II procollagen mRNA has been partially purified from embryonic chick sternal cartilage by guanidine hydrochloride extraction, sucrose gradient sedimentation and Sepharose 4B chromatography. Double stranded cDNA was synthesized using AMV reverse transcriptase and E. coli DNA polymerase I, tailed using terminal transferase and inserted into the Pst I site of pBR322. Two putative type II procollagen cDNA clones have been characterized. Both plasmids hybridize to 2 sternal RNA species, a major species of 5.3 kb and a minor species of 7 kb. These RNAs are present in total RNA from sterna and differentiated limb bud cultures but are not detected in RNA from stage 24 limb bud which has not yet differentiated to cartilage or in RNA from calvaria. The time of appearance of these RNAs during the differentiation of limb mesenchyme in culture parallels the appearance of translatable type II procollagen mRNA.

A recombinant human DNA library was screened using probes corresponding to the chick alpha 1 (II) procollagen gene. This resulted in the isolation of 2 different genomic clones, LgHCol(II)a and LgHCol(II)b. LgHCol(II)a was identified as corresponding to the alpha 1(II) gene by comparative hybridization and DNA sequence analysis. DNA sequence established that LgHCol(II)a extends at least from amino acid 694 of the triple helix through 54 amino acids of the COOH-propeptide. Hybridization with a probe containing only the exon at the 3' end of the chicken gene suggests that the clone contains the 3' end of the human gene. Thus LgHCol(II)a contains approximately 40% of the coding sequences of the human type II collagen gene.

In summary, the exon/intron structure of the chicken type II collagen gene is identical with that of the chicken alpha 2(I) collagen gene and differs at only one known position from the human and mouse alpha 1(I) genes. However, the chicken type II gene is different from the chicken alpha 2(I) gene in that it is considerably shorter because of a much smaller average intron size and in that the G+C composition of the introns is much higher. The codon usage of the type II genes also shows characteristic differences. There is a single copy of the chick type II gene per haploid genome.

Messenger RNAs for the alpha 1 and alpha 2 chains of type I procollagen were partially purified from total embryonic chicken calvaria using gel chromatography on Sepharose 4B and used to construct recombinant cDNA clones corresponding to both mRNAs. Restriction site mapping, nucleotide sequencing and hybridization to RNA blots were used to show that clones pCAL1 and pCAL2 contain inserted sequences corresponding to the mRNAs for chicken alpha 1 and alpha 2 procollagen chains, respectively.

The copy number of the type II procollagen gene has been determined by quantitative hybridization of cloned DNA to Southern blots of total cellular DNA from chicken. Two different DNA probes have been used. One contains the 289 base pair exon coding for the junction between the triple helical domain and the carboxyl-terminal propeptide and the other contains an 162 base pair exon coding for amino acids 712-765 of the triple helical domain. Both fragments hybridize to single bands present at one copy per haploid genome in genomic DNA from chicken liver or sternal cartilage after these DNAs are cleaved with either HinfI of PstI. When the hybridization stringency and washing conditions were modified sufficiently to stabilize hybridization of heteroduplexes with up to 31% mismatch, no additional hybridizing fragments were detected. These results show that there is not a second copy of this gene or a pseudogene which is more closely related to the type II procollagen gene than the alpha 1 type I procollagen gene. These studies suggest that the cartilage 3 alpha collagen chain, which appears to be very similar to the alpha 1 (II) collagen chain, is encoded by the type II procollagen gene but is differently processed than the major type II gene product, and that the alpha 1 (II) Minor chain identified in bovine type II collagen preparations most likely represents an allelic form of type II collagen rather than a product of a different gene.

The DNA sequence of two overlapping cDNA clones and a genomic lambda clone covering the region coding for 288 amino acids at the COOH terminus of the chicken type II procollagen gene is reported. This region consists of 4 exons coding for the last 15 amino acids of the triple helical domain and 273 amino acids which correspond to the COOH-terminal telopeptide and COOH-terminal propeptide. The sequence, base composition, and codon usage of this region of the type II procollagen gene show particularly high similarity to those of the chicken alpha 1(I) procollagen gene and differ from those of the alpha 2(I) and alpha 1(III) gene sequences. Two DNA tracts of low sequence similarity were observed. One of these regions spans the telopeptide and COOH-terminal propeptidase cleavage site, although 4-5 amino acids at the actual cleavage site are conserved compared with the alpha 1(I) and alpha 2(I) genes. A region of unusually high nucleotide sequence conservation is present in exon 2 (amino acids 171c - 186c ) consisting of approximately 45 nucleotides with only one or two base substitutions compared with the other procollagen genes. Within this conserved sequence is a site for carbohydrate attachment. The 3' nontranslated sequence of the type II procollagen mRNA is longer than that of either the alpha 1(I) or alpha 2(I) mRNA and contains several unusual long tracts consisting primarily of one or two bases. Although the canonical site for polyadenylation is not present, two related sequences, AACAAA and ATATAAA , are present 32 and 41 bases preceding the end of the major RNA species. The exon/intron structure of the type II procollagen gene is similar to that of other collagen genes which have been described. This DNA sequence provides the first extensive report of the amino acid sequence of chicken type II procollagen.

A single-stranded DNA probe complementary to chicken type II procollagen mRNA has been used to quantitate levels of that mRNA present in chicken limb mesenchyme during cartilage differentiation. Excess labeled probe prepared from a cDNA template cloned in M13mp9 was hybridized to completion to increasing amounts of total RNA and assayed by protection from S1 nuclease digestion. Estimates of the absolute levels of type II procollagen RNA were determined using the M13mp9 template containing the coding strand as a standard. RNA complementary to the probe increased from 20 copies per diploid genome in stage 24 limb to approximately 2000 copies per diploid genome in stage 24 limb mesenchyme which had differentiated to cartilage in culture. Similar levels were found in cartilage from stage 31 limb. Sternal cartilage from 17-day embryos contained approximately 10,000 copies per diploid genome suggesting that the level of expression of this gene is different in limb growth cartilage compared with sternal cartilage. Low but detectable levels of RNA complementary to the probe were observed in limb at stages 20-24. Since a large fraction of the type II procollagen RNA in these early limbs is associated with polysomes, the type II procollagen gene appears to be expressed at a low level prior to phenotypic differentiation and prior to the accumulation of immunologically detectable levels of type II collagen.

Overlapping genomic clones have been isolated that contain the alpha chain and COOH-terminal propeptide coding regions of the chicken type II procollagen gene. All type II procollagen exon sequences present in these clones have been identified and mapped by DNA sequencing. These include 43 exons coding for the alpha-chain triple helix, 1 exon coding for the junction between the COOH-terminal propeptide and the alpha-chain region, and 3 exons coding for the COOH-terminal propeptide and 3' noncoding sequences. With the exception of one additional intron between 2 exons coding for amino acids 568-585 and 586-603, exon-intron boundaries have been conserved when compared with genes for all other characterized genes for fibrillar collagens. The chicken type II procollagen gene differs from most other collagen genes in having introns of considerably smaller average size. The size distribution of the introns suggests that approximately equal to 80 base pairs may be a minimal functional size for introns in this gene. This size of intron may be necessary in a gene with a very large number of small exons to prevent aberrant splicing from removing exon sequence together with intron sequence.

We examined the osteoblastic phenotype of permanently transfected ROS 17/2.8 cells in culture and in vivo, in order to evaluate their relevance for studies of the regulation of gene expression and gene function in osteoblastic cells. Recent reports indicate that the progeny transfected cells may substantially vary and differ from the parental cell line in their phenotype, particularly in their tumorigenicity. ROS 17/2.8 cells were transfected with genetic constructs expressing the CAT gene from either the rat alpha 1 (I) or the mouse alpha 2 (I) collagen promoters. Forty-four clonal cell lines display a range of CAT expression from the transfected collagen promoters in culture. Four of these cell lines were further characterized. Alkaline phosphatase activity in these four cell lines is higher than in fibroblastic cells. These four cell lines are tumorigenic in immunocompatible ACI rats and form calcified tumors similar to those formed by ROS 17/2.8. CAT expression could be demonstrated in tumor extracts of two of the four cell lines, which also expressed higher CAT levels in culture. We conclude that permanently transfected ROS 17/2.8 derived cell lines maintain their tumorigenicity and their osteoblastic-like phenotype, and thus may provide a useful system for studies of gene function and regulation in osteoblast-like cells and bone-like tissue in vivo.

A critical event in limb cartilage differentiation is a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed and interact with one another prior to initiating cartilage matrix deposition. Fibronectin (FN) has been suggested to be involved in regulating the onset of condensation and chondrogenesis by actively promoting prechondrogenic aggregate formation during the process. We have performed a systematic quantitative study of the expression of the FN gene during the progression of chondrogenesis in vitro and in vivo. In high-density micromass cultures of limb mesenchymal cells, FN mRNA levels increase about 5-fold coincident with the crucial condensation process, and remain relatively high during the initial deposition of cartilage matrix by the cells. Thereafter, FN mRNA levels progressively decline to relatively low levels as the cultures form a virtually uniform mass of cartilage. The changes in FN mRNA levels in vitro are paralleled closely by changes in the relative rate of FN synthesis as determined by pulse-labeling and immunoprecipitation analysis. The relative rate of FN synthesis increases 4- to 5-fold at condensation and the onset of chondrogenesis, after which it progressively declines to low levels as cartilage matrix accumulates. High levels of FN gene expression also occur at the onset of chondrogenesis in vivo. In the proximal central core regions of the limb bud in which condensation and cartilage matrix deposition are being initiated, FN mRNA levels and the relative rates of FN synthesis become progressively about 4-fold higher than in the distal subridge region, which consists of undifferentiated mesenchymal cells that have not yet initiated condensation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10-15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.

As limb mesenchymal cells differentiate into chondrocytes they initiate the synthesis of a cartilage-specific sulfated proteoglycan, cartilage-characteristic type II collagen, and other cartilage-specific proteins. In the present study, in situ hybridization with a 32P-labeled cloned cDNA probe complementary to mRNA encoding the core protein of cartilage proteoglycan has been used to visualize and localize the accumulation of cartilage proteoglycan core protein mRNA sequences during development of the chick limb bud in vivo. When the probe was hybridized to sections through 7-day (stage 32) limbs, an intense hybridization signal was observed over the well-differentiated cartilage rudiments of the limb, while no signal above background was observed over nonchondrogenic tissues including muscle, loose connective tissue, and epidermis. At early stages of limb development, an accumulation of silver grains representing hybridizable core protein mRNA first became detectable in the proximal central core of the limb where the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation was occurring. In fact, the pattern of silver grain accumulation closely followed the pattern of mesenchymal cell condensation, and no hybridizable core protein mRNA sequences were detectable in the limb bud prior to condensation. Cartilage-characteristic type II collagen mRNA was colocalized with core protein mRNA in the condensing central core of the limb suggesting that the genes for these two major constituents of cartilage matrix are coordinately regulated at the onset of chondrogenesis. Furthermore, the appearance of hybridizable core protein mRNA was closely followed by the appearance of the protein for which it codes as detected by immunohistochemical staining with monospecific antibody. These observations support the hypothesis that at the initial stages of limb chondrogenesis core protein gene expression is controlled primarily at the transcriptional level.

Mitochondrial DNA (mtDNA) from sheep and goat was compared by restriction endonuclease analysis and heteroduplex mapping in the electron microscope. The fragment patterns produced by endonuclease Hae III from three individual sheep and two goat mtDNAs all differed from each other. The three sheep mtDNAs had identical Eco RI and Hind III fragments, but the two goat mtDNA patterns differed from each other as well as from sheep mtDNA. We estimate that each sheep mtDNA differs from each other by 0.5-1% of its nucleotide sequences, the two goat mtDNAs by 1-2%, and there is a 6-11% sequence difference between sheep and goat mtDNAs. We have mapped the Eco RI and Hind III sites of goat and sheep mtDNA and determined the positions of the D loop, which marks the replication origin, relative to the restriction map. The D loops are at homologous positions on the mtDNAs from both species, but the goat D loop is only 75% as long as the sheep D loop. Regions with a high degree of sequence divergence occur at both ends of the D loop. We suggest that a duplication of about 150 base pairs has occurred in the region where the sheep and goat D loops differ in length. We discuss mtDNA evolution in terms of divergence of isolated "mitochondrial DNA clones."

Equations for the measurement of the supertwisting of circular DNA's from banding positions in buoyant density gradients containing intercalating dyes have required the use of SV40 DNA isolated from virions as a reference DNA. These equations are modified to allow the use of any closed circular DNA of known superhelix density as a reference DNA.

Type II collagen is a major component of cartilage extracellular matrix. Differentiation of mesenchyme into cartilage involves the cessation of type I collagen synthesis and the onset of type II collagen synthesis. Solution hybridization of mRNA isolated from chick limb buds with a cDNA probe to type II collagen mRNA showed the presence of small amounts of type II collagen message in mesenchymal chick limbs. We have examined the localization of type II collagen mRNA in mesenchymal chick wing buds by in situ hybridization using single stranded RNA probes. Our results show a small but detectable amount of type II collagen RNA distributed uniformly in early limbs until the first precartilage condensations form at stage 22. This is interesting because it is known that mesenchyme isolated from chick wing buds has the capacity to undergo chondrogenesis in culture, even if taken from nonchondrogenic areas of the limb. At stage 23, type II collagen mRNA is found at significantly increased levels in the cells of the precartilage condensation when compared to the other limb cells. As chondrogenesis proceeds, the amount of type II collagen RNA increases even more in cells of the cartilage elements. The signal in the peripheral tissue is indistinguishable from background. These results show that type II collagen message exists at low levels in cells throughout the mesenchymal chick wing bud, until the formation of the condensation results in an elevation of type II mRNA in the prechondrogenic cells found in the core of the limb.

Total RNA, prepared from chicken limb bud cultures undergoing differentiation to cartilage, has been translated in a wheat germ cell-free protein-synthesizing system. Antibodies against chondroitin sulfate proteoglycan core protein immunoprecipitate a single component which migrates as a protein of 340,000 daltons in sodium dodecyl sulfate/polyacrylamide gels. The messenger RNA for this protein sediments at approximately 27 S in 70% formamide or aqueous sucrose gradients. The 340,000-dalton protein is present in cell-free translation products directed by RNA prepared from limb bud cultures and sternae and is absent in cell-free translation directed by RNA prepared from embryonic calvaria or liver. The level of synthesis of this protein is greatly reduced when RNA prepared from limb bud cultures inhibited from differentiation by BrdUrd is used. (Pre)pro alpha 1(I), -alpha 2(I), and -alpha 1(II) collagen bands have been identified on gels by electrophoresis of collagenase-digested or immunoprecipitated cell-free translation products directed by RNA from differentiating limb bud cultures, embryonic sternae, and embryonic calvaria.

An estimation of the DNA sequence divergence between defined DNA secquences of individuals or species may be made from comparison by gel electrophoresis of restriction endonuclease digests. This analysis is applicable to purified DNA sequence of moderate complexity (1-100 X 10(6) daltons) which have diverged by base substitution of 0.5 to 25% of nucleotides.

The separation between open and closed circular DNA in buoyant CsCl gradients containing intercalating dyes depends on the superhelix density of the closed form. These separations are about 1.8 times larger with propidium iodide than with ethidium bromide. The superhelix densities of mitochondrial DNA from HeLa cells and Lytechinus pictus eggs appear to be about two thirds that of mitochondrial DNA from rat and rabbit liver.