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Basic fibroblast growth factor (bFGF), a member of the fibroblast growth factor family, potently induces increased vascular smooth muscle cell (SMC) proliferation and decreased expression of type I collagen. Recently, our laboratory demonstrated that, in bovine vascular SMCs, expression of B-myb, a member of the myb gene family, is dependent upon cellular growth state and that B-myb decreases alpha1(I) collagen promoter activity in transient transfection assays. Nuclear run-off analysis indicated that the decrease in alpha1(I) collagen mRNA level seen upon bFGF treatment was due to a decline in the rate of alpha1(I) procollagen gene transcription. Thus, we investigated the potential role of B-Myb in the down-regulation of type I collagen gene expression by bFGF. Using Northern blot analysis, we found that bFGF treatment of bovine aortic SMCs caused an increase in B-myb mRNA levels. Ectopic expression of B-myb decreased endogenous alpha1(I) collagen mRNA levels. Importantly, introduction of a B-myb antisense oligonucleotide prevented the drop in the alpha1(I) collagen mRNA levels seen upon treatment with bFGF. Together, these results indicate that B-myb mediates signals leading to the decreased rate of alpha1(I) collagen gene transcription caused by bFGF.

In previous work, we delineated a proximal region of the human alpha1(I) collagen gene (COL1A1) promoter necessary to direct its basal transcription in fibroblasts. This region has potential recognition sites for a variety of DNA-binding proteins. Here, we show that the -129/-107-bp sequence in this region of the promoter, which harbors an inverted CCAAT motif closely linked to a GC-rich direct repeat and is perfectly conserved between mouse and human, specifically bound the transcription factors Sp1, Sp3, and CTF/NF-1 in nuclear extracts from human skin and lung fibroblasts. Drosophila Schneider L2 cells lacking endogenous Sp activity were used to investigate the effect of expression of Sp or CTF/NF-1 transcription factors on COLlAl promoter activity. Expression of Sp1 caused potent trans-activation of a COL1A1 promoter (-174 to +42bp). In contrast, expression of Sp3, which binds to the same recognition sites as Sp1, and CTF/NF-1 stimulated COL1A1 promoter activity only at higher concentrations, and Sp2 did not transactivate. Expression of a 10-fold excess of Sp3, but not CTF/NF-1 or Sp2, abrogated the stimulation of COL1A1 promoter activity induced by Sp1. TGF-beta at concentrations previously shown to increase COL1A1 transcription caused a decrease in the relative amount of Sp3 in fibroblast nuclear extracts. These results suggest that both Sp1 and Sp3 bind to the proximal COL1A1 promoter and stimulate its activity; however, their interaction with each other may result in repression of Sp1-induced COL1A1 transcription. Alterations in the relative amounts or DNA-binding activities of these transcription factors in a cell- or signal-specific manner may contribute to the control of transcription from the COL1A1 promoter. The present findings, and recent observations implicating Sp1 and its homologs in regulating the expression of several collagen genes, suggest that the family of Sp1 transcription factors play a role in physiological and pathological modulation of connective tissue accumulation.

Type XI collagen, a fibril-forming collagen, is important for the integrity and development of the skeleton because mutations in the genes encoding its consituent alpha chains have been found in some osteochondrodysplasias. We provide data that complete information for the coding sequence of human alpha 2(XI) procollagen, with details of the promoter region and intron-exon organization at the 5' and 3' ends of the gene (COL11A2), including the transcription start and polyadenylation sites. COL11A2 is 30.5 kb with a minimum of 62 exons, differing from other reported fibrillar collagen genes because the amino propeptide is encoded by 14 not 5 to 8 exons. But exon numbers for the carboxy propeptide and 3'-untranslated region are conserved. The promoter region of COL11A2 lacks a TATA box but is GC-rich with two potential SP1 binding sites. Mouse alpha 2(XI) collagen mRNAs undergo complex alternative splicing involving three amino-terminal propeptide exons but only one of these has been reported for COL11A2. We have located these missing human exons and have identified splice signals that point to additional splice variants. We have precisely mapped COL11A2 within the major histocompatibility complex on chromosome 6. The retinoid X receptor beta (RXR beta) gene is located 1.1 kb upstream of COL11A2. KE5, previously thought to be a distinct transcribed gene sequence, was mapped within COL11A2 in the alternatively spliced region, raising the question whether KE5 and COL11A2 are separate genes.

Objective: To define the clinical, pathological and molecular genetic characteristics of a family with mild spondyloepiphyseal dysplasia (SED) and precocious osteoarthritis., Methods: The proband was a 46-year-old man with precocious generalized OA, tall stature, mild chondrodysplasia and moderate deafness. His daughter, aged 21, showed similar clinical features. Electron microscopic (EM) analysis of collagen from an affected joint of the proband was performed. DNA was extracted from whole blood on the proband, his affected daughter, unaffected wife and second daughter, to look for a mutation in exons 31 or 11, sites where point mutations have been previously described in mild forms of SED. After finding no mutation in exon 31, exon 11 of COL2A1 was further analyzed. Exon 11 was amplified using polymerase chain reaction (PCR), and screening for the mutation was undertaken using a restriction enzyme digestion, the recognition sequence of which is altered by this point mutation. Sequence analysis was then performed., Results: Electron microscopic (EM) analysis of cartilage from the proband showed thin appearing collagen fibrils organized into parallel lamellar structures. DNA studies revealed a single base change in one allele of exon 11 which produced an arginine to cysteine mutation at position 75 of the triple helix of type II collagen in the proband and his affected daughter., Conclusion: This is the 2nd example of an Arginine75-Cysteine mutation associated with SED; in our case, however, contrasting clinical features were present. Recurrent mutations at a few specific sites of COL2A1 suggest the possibility of susceptibility "hot spots" for mutational events.

Objective: To investigate the occurrence and clinical correlation of the arg519-to-cys mutation in the type II procollagen gene in patients with osteoarthritis (OA)., Methods: Sixty-six subjects from 7 families with a strong family history of generalized OA and 13 patients with erosive OA were evaluated clinically and radiologically. Blood samples from 58 subjects in the familial OA group and from all 13 patients with erosive OA were obtained for DNA analysis. Exon 31 of COL2A1, which spans residue 519, was amplified by polymerase chain reaction., Results: The arg519-to-cys mutation was detected in 2 of the 7 families with generalized OA. In these 2 families, the mutation was present in the 2 probands and in 19 other clinically affected family members, as well as in 3 (so-far) clinically unaffected family members (ages 25, 14, and 11 years). It was absent in 18 clinically unaffected members tested. The mutation was associated with a distinctive pattern of early-onset, aggressive, generalized OA with a mild spinal chondrodysplasia. Inheritance was autosomal dominant. No mutation was found in any of the patients with erosive OA., Conclusion: The arg519-to-cys mutation defines a new pathogenic factor in generalized OA with characteristic clinical and radiologic features. The demonstration of a mutation in 3 of 8 families with OA studied thus far suggests a significant incidence of genetically related clinical OA.

Previous reports have provided inconsistent data as to the cis-regulatory elements that are essential for correct expression of the gene for the pro alpha 1 (I) chain of type I procollagen (COL1A1) in the many tissues in which the protein is synthesized. Here, two internally deleted minigene versions of the human COL1A1 gene were used to prepare transgenic mice. The constructs made it possible to test regulatory sequences in the normal context of the gene. Also, in contrast to the reporter genes used in previous experiments, the constructs made it possible to assay quantitatively expression of the exogenous genes relative to expression of the endogenous COL1A1 gene, both as mRNA and as protein. The average level of expression of the minigenes varied among three transgenic lines, but the ratio of expression of the minigenes to expression of the endogenous gene was the same in all transgenic mice of a given line. Within the same line, the ratio of expression was essentially the same in nine or more tissues in which expression of the endogenous gene varied widely. Also, the ratio of expression within a given line was the same in 15-day-old embryos and in mice ranging in age from 4 days to 4 months. In addition, the ratio remained constant during repair of a surgical wound. The results demonstrated, therefore, that the minigene constructs with about 2.3 kb of the promoter region and about 2 kb of the 3'-flanking region contained all of the sequences necessary for correct expression of the genes in a tissue-specific and development-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular defect responsible for a case of mild osteogenesis imperfecta (OI) with repeated femoral fractures was investigated. The proband and his mother, who presented minor OI signs but no bone fractures, were shown to produce normal and abnormal type-I procollagen molecules in their dermal fibroblasts. The molecular defect was localized in about half of the proband's pro alpha 1(I) mRNA molecules by chemical cleavage with piperidine of hydroxylamine-reacted mRNA:cDNA heteroduplexes. The corresponding region was reverse-transcribed and amplified by polymerase chain reaction (PCR). Cloning and sequencing of the amplified products revealed in both subjects a G-to-A transition in the first base of codon 901 of the alpha 1(I) triple helical domain, which led to a serine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified genomic DNA from fibroblasts and leukocytes confirmed the heterozygous nature of both patients and proved the absence of mosaicism. The presence of the mutation was excluded in other healthy family members, who were reported to have bluish selerae. The mild phenotypic outcome of this newly characterized mutation contradicts previous findings on glycine substitutions in the C-terminal region of collagen triple helix, most of which caused lethal OI.

Linkage analysis with restriction fragment length polymorphisms for the gene for type II procollagen (COL2A1) was carried out in a family with the Stickler syndrome, or arthro-ophthalmopathy, an autosomal dominant disorder that affects the eyes, ears, joints, and skeleton. The analysis demonstrated linkage of the disease and COL2A1 with a logarithm-of-odds score of 1.51 at zero recombination. A newly developed procedure for preparing cosmid clones was employed to isolate the allele for type II procollagen that was linked to the disease. Analysis of over 7000 nucleotides of the gene revealed a single base mutation that altered a CG dinucleotide and converted the codon CGA for arginine at amino acid position alpha 1-732 to TGA, a stop codon. From previous work on procollagen biosynthesis, it is apparent that the truncated polypeptide synthesized from an allele with a stop codon at alpha 1-732 cannot participate in the assembly of type II procollagen, and therefore that the mutation would decrease synthesis of type II procollagen. It was not apparent, however, why the mutation produced marked changes in the eye, which contains only small amounts of type II collagen, but relatively mild effects on the many cartilaginous structures of the body that are rich in the same protein.

The nucleotide sequence of the human procollagen alpha 1(II) collagen gene extending from within the first intron through exon 15, and part of the 15th intron has been determined. This sequence analysis (7056 bases) identifies the intron/exon organization of the region of this gene encoding the N-propeptide and part of the triple-helical domain. Structural comparison of this with the genes of other human fibrillar collagens shows considerable diversity in terms of size and number of introns and exons that encodes the N-propeptide domain. Although the genomic structure of the human procollagen alpha 1(II) gene is quite different from the rat procollagen alpha 1(II) gene, the nucleotide coding sequences are 89% identical.

A high frequency restriction fragment length polymorphism (RFLP) at the 3'-end of the pigeon pro alpha 2(1) collagen gene was detected using the restriction endonuclease EcoR1. The distribution of this allelic variant was analyzed in DNA isolated from White Carneau pigeons genetically susceptible to the development of spontaneous atherosclerosis. The atherogenic phenotype in individual pigeons was measured by the determination of total cholesterol and cholesterol ester levels in the celiac focus of the thoracic aorta of adult White Carneau pigeons. Aortic wall cholesterol levels correlated with an increase in lesion size. No correlation, however, was observed between allelic variants of the pigeon pro alpha 2(1) collagen gene and the atherogenic phenotype in White Carneau pigeons suggesting lack of linkage between this allelic marker and the genetic susceptibility to spontaneous atherogenesis. This is the first study of its kind in this animal model and serves to provide a basis for the further analysis of co-segregation of RFLPS in candidate genes to this polygenic phenotype.

Tubulointerstitial changes in the diabetic kidney correlate closely with the decline in glomerular filtration. In this study, we used a cell culture system of mouse proximal tubule epithelial cells to test the effects of glucose on cell growth, size, and matrix biosynthesis. [3H]thymidine incorporation was significantly inhibited in cells grown in 450 mg/dl glucose, compared with cells grown in 100 mg/dl glucose. The cells grown in the higher glucose concentration were slightly larger, their protein content and the total protein synthetic rate were significantly increased, and they secreted approximately twice as much procollagens type IV and type I. Concordantly, steady-state procollagen mRNA levels were also increased: 2.6-fold for the alpha 1(IV) and 2.2-fold for the alpha 2(I) procollagens. Additionally, nuclear run-off studies demonstrated that procollagen gene transcription rate was stimulated approximately 50%; beta-actin transcription rate was not altered. We used chloramphenicol acetyltransferase (CAT) reporter gene constructs to determine whether the increased transcription rate of alpha 2(I) gene was associated with activation of its enhancer sequence. Cells transfected with the enhancer demonstrated more than fivefold increase in CAT activity when cultured in the high-glucose medium. These studies demonstrate a multitude of effects of high ambient glucose concentrations on proximal tubule cell growth and collagen biosynthesis; cell proliferation is decreased although cell hypertrophy occurs. Procollagen gene transcription rate is stimulated and this response contributes to the observed increase in procollagen mRNA content. Activation of an enhancer sequence may be one possible mode through which high glucose levels increase the transcription of procollagen type I, presumably involving trans-acting factor(s).

A splicing mutation in the pro-alpha 2(I) collagen gene of a patient with Ehlers-Danlos syndrome type VII has been characterized. Protein microsequencing of peptides prepared from the patient's dermal collagen identified an interstitial deletion of 18 residues. The deleted segment corresponds to the amino-terminal telopeptide junction domain encoded by the sixth exon of the pro-alpha 2(I) collagen gene. Sequencing of specifically primed cDNA clones confirmed the presence of two distinct populations of pro-alpha 2(I) mRNAs, a normal one and another which lacks the sequences of exon 6. Limited sequencing of genomic clones showed that one of the pro-alpha 2(I) alleles displays a conservative change in the seventh codon of exon 6 (GAC for GAT), and a base substitution at position +1 of intron 6 (A for G). Since the normal transcript contains the GAT codon, the intronic change was associated with the allele that gives rise to the shortened pro-alpha 2(I) collagen mRNA. The two allelic fragments were subcloned into an expression vector and the pattern of splice-site selection for exons 5-8 was assessed for each of the constructs after transfection into COS cells. This documented skipping of exon 6 sequences only in transcripts of the minigene construct that harbors the G to A transition. Expression of allelic cross-constructs confirmed that the single-base substitution at position +1 of intron 6 is the mutation responsible for the abnormal joining of exons 5 and 7 sequences in the patient's shortened pro-alpha 2(I)mRNA.

The complete DNA sequence (6 kb) of the 5' portion of the human type II procollagen gene (COL2A1) was determined from the promoter through the third exon including intron sequences and 690 bp of 5' flanking sequence. Three regions between -501 and -649 in the human promoter share high sequence homology to the rat type II procollagen gene and suggest that the functional sequences within the promoter may extend to at least 649 bp upstream from the start site of transcription. The first intron of the human gene contains elements known to play a role in transcription of other genes: three GC boxes, an inverted repeat with homology to a serum responsive element, a viral core enhancer motif, a high-affinity recognition sequence for nuclear factor-1, and an alternating purine/pyrimidine stretch composed of GT repeats. Both the promoter and a portion of the first intron have a high percentage of G + C residues and a high frequency of CpG dinucleotides. In addition, a protein domain that has been identified in the human COL2A1 gene is present in pro-alpha 1(I) and pro-alpha 1(III) collagen but was not previously described for pro-alpha 1(II) collagen. On the basis of this new information we present a modified gene structure for the exons encoding the pro-alpha 1(II) collagen NH2-propeptide.

A cosmid clone was isolated that contained an allele for the type II procollagen gene previously shown to be coinherited with primary generalized osteoarthritis in a large family. Affected members of the family had evidence of a mild chondrodysplasia, but they developed progressive osteoarthritic changes in many joints that had no epiphyseal deformities. The clone contained 52 of the 54 exons of the gene. Nucleotide sequencing of greater than 20,000 base pairs from the clone demonstrated that all the coding sequences and all the intron-exon boundaries were normal except for a single base mutation that converted the codon for arginine at position 519 of the alpha 1(II) chain to a codon for cysteine, an amino acid not found in type II collagen from humans or a variety of other species. The mutation was found in all affected members of the family but not in unaffected members or in 57 unrelated individuals.

The 3500-base pair region located immediately upstream of the transcriptional start site of the human pro-alpha 2(I) collagen gene contains all the sequences necessary for cell-specific transcription. In transient expression assays, the pro-alpha 2(I) collagen promoter directed the production of high levels of bacterial chloramphenicol acetyltransferase in collagen-producing human fetal fibroblasts. Enzyme activity, on the other hand, was nearly undetectable in extracts from collagen-nonproducing immortalized lymphoblasts. Deletion experiments narrowed the active segment of the human promoter to a phylogenetically conserved sequence comprised between nucleotides-376 and -108, relative to the initiation site of transcription. In similar analyses, the pro-alpha 1(I) collagen gene failed to direct cell-specific transcription. As part of this study, the controversial issue surrounding the putative enhancer element in the first intron of the human pro-alpha 1(I) collagen gene also has been reconsidered. Accordingly, we now propose a more restricted definition of this cis-acting DNA element since its action is exerted in an orientation-preferred manner and with a strong specificity for its own promoter. Moreover, stimulation does not appear to be tissue-specific. Finally, evidence is presented supporting the notion that although structurally different and distinctly arranged, the regulatory sequences of the type I collagen genes may bind similar trans-acting factors.

Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, P1-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine pro alpha 1(I) collagen (Col1a1) gene. These mutant constructs have been transfected into homozygous Mov13 fibroblasts that do not express the endogenous Col1a1 gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Ile (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for Ile (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for Ile at position 776. The wild-type alpha 2(I) chains derived from the endogenous Col1a2 gene were also resistant to enzyme digestion when they were complexed with the mutant alpha 1(I) chains, indicating that the presence of normal alpha 1(I) sequences is critical for cleavage of the alpha 2(I) chains in the type I heterotrimer.

Identical G+1 mutations in three different introns of the gene for type III procollagen (COL3A1) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome. Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing. The G+1 to A mutation in intron 16 caused extensive exon skipping, the G+1 to A mutation in intron 20 caused both use of a cryptic splice site and retention of all the intron sequences, and the G+1 to A mutation in intron 42 caused efficient use of a single cryptic splice site. The different patterns of RNA splicing were not explained by evaluation of potential cryptic splice sites in the introns by either their homology with 5'-splice sites from other genes or by their delta G(0)37 values for binding to U1 RNA. Instead, the results suggested that the patterns of aberrant RNA splicing were primarily determined by the relative rates at which adjacent introns were normally spliced.

We have cloned and characterized several overlapping cDNAs that specify a large portion of a Paracentrotus lividus fibrillar collagen molecule. Our conclusions are based on sequencing data, which showed that the clones code for a 786-amino acid collagenous domain composed of an uninterrupted series of Gly-X-Y repeats and for a 265-amino acid carboxyl-terminal globular extension. The latter domain exhibits features highly reminiscent of those of the vertebrate counterparts, notably a putative carboxyl-peptidase cleavage site, a series of similarly arranged cysteinyl residues, and an N-linked glycosylation attachment site. In situ and Northern blot hybridizations have established the size, time of appearance, and tissue localization of the collagen mRNA during sea urchin development. The collagen transcript, 9 kilobases in length, is first detected in the primary and, more predominantly, in the secondary mesenchyme cells of late gastrulae where it progressively accumulates thereafter. This and other work (D'Alessio, M., Ramirez, F., Suzuki, H.R., Solursh, M., and Gambino, R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9303-9307) provide evidence of a genetic heterogeneity of fibrillar collagens in the sea urchin embryo and suggest that the two genes are activated in the same cell lineages at distinct developmental stages.

We have determined the nucleotide sequence of several overlapping cDNA clones encoding the amino-terminal portion of human alpha 1(XI) procollagen. These experiments have revealed that this domain of the pro-alpha(XI) chain displays structural features common to other fibrillar procollagen molecules, such as a putative amino-terminal proteinase cleavage site and an interrupted collagenous segment. In the latter, structural similarities were noted when alpha 1(XI) was compared with alpha 1(II) and alpha 2(V) procollagens. Overall, however, the amino-terminal region of pro-alpha 1(XI) differs greatly in composition and size from that of other fibrillar chains. Nearly three-fourths of this domain is in fact composed of a 383-amino acid globular region in which a 3-cysteine cluster signals the transition to a long and highly acidic carboxyl-terminal segment. Finally, the unrestricted expression of this cartilage-specific collagen gene has been confirmed by the finding of high levels of pro-alpha 1(XI) mRNA in two human rhabdomyosarcoma cell lines.

To investigate whether the human pro alpha 1(I) collagen chain could form an in vivo functional interspecies heterotrimer with the mouse pro alpha 2(I) collagen chain, we introduced the human COL1A1 gene into Mov13 mice which have a functional deletion of the endogenous COL1A1 gene. Transgenic mouse strains (HucI and HucII) carrying the human COL1A1 gene were first generated by microinjecting the COL1A1 gene into wild-type mouse embryos. Genetic evidence indicated that the transgene in the HucI strain was closely linked to the endogenous mouse COL1A1 gene and was X linked in the HucII transgenic strain. Northern (RNA) blot and S1 protection analyses showed that the transgene was expressed in the appropriate tissue-specific manner and as efficiently as the endogenous COL1A1 gene. HucII mice were crossed with Mov13 mice to transfer the human transgene into the mutant strain. Whereas homozygous Mov13 embryos die between days 13 and 14 of gestation, the presence of the transgene permitted apparently normal development of the mutant embryos to birth. This indicated that the mouse-human interspecies collagen I heterotrimer was functional in the animal. The rescue was, however, only partial, as all homozygotes died within 36 h after delivery, with signs of internal bleeding. This could have been due to a functional defect in the interspecies hybrid collagen. Extensive analysis failed to reveal any biochemical or morphological abnormalities of the collagen I molecules in Mov13-HucII embryos. This may indicate that there was a subtle functional defect of the interspecies hybrid protein which was not revealed by our analysis or that another gene has been mutated by the retroviral insertion in the Mov13 mutant strain.

A stability map of local secondary structure of the mRNA of the triple-helical alpha 2(I) chain domain of chicken type I collagen was obtained by plotting the free energy of the optimal secondary structure of a local segment in mRNA against the segment position along a base sequence of the mRNA. It was found that the positions of the minima of free energy in the plot coincide with the positions where synthesis pauses of the alpha-chain polypeptides of the corresponding sizes translated from the mRNA have been reported to occur (1). The codon usage pattern of each of the three major amino acids of the alpha-chain domain of the collagen, Gly, Pro and Ala, fluctuates considerably along the base sequence segments of the mRNA and a deviation of the pattern from that of the average of the whole alpha 2(I) chain domain mRNA, particularly for Gly codons, leads to a loss of the stability of the local secondary structure of the mRNA. The results suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the mRNA of the chicken collagen alpha 2(I) chain domain which leads to a nonuniform polypeptide elongation pattern.

Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.

A new procedure for the synthesis of double stranded cDNA, based upon release of mRNA by "in vitro" translation, was used to clone type IV collagen. Collagen synthesizing polysomes selectively isolated from a mouse parietal yolk sac carcinoma (PYS-2) were used for translation in an heterologous cell-free system. Translation products were collagenase-sensitive and displayed an electrophoretic mobility correspondent to type IV collagen. Translation released mRNA was employed to construct a 100 base pairs long cDNA clone which hybridized to a 7,800 nucleotides long mRNA. Peptides synthesized by "in vitro" translation of hybrid selected mRNA displayed an electrophoretic mobility compatible with that of alpha 1 (IV) collagen, were sensitive to collagenase and were immunoprecipitated by anti-type IV collagen antibody.

Restriction fragment length polymorphisms (RFLPs) in types I and III procollagen genes were studied in 62 scleroderma patients and 138 healthy controls. Allelic frequencies were determined for each RFLP, and comparisons were made between the 2 populations, stratifying them by race when appropriate. No statistically significant differences were observed for the frequencies of any of the RFLPs studied.

Transformation of the human embryonic lung fibroblast line, WI-38, with simian virus 40 (SV40) results in inactivation of the type I procollagen genes. No type I collagen or procollagen mRNA is detected in these transformed cells, as determined by polyacrylamide gel electrophoresis. Analysis of the methylation patterns of these genes showed the type I procollagen genes to be hypermethylated at certain cytosine residues in the transformed cells. However, several of the cytosine residues were methylated in the normal cells where these genes are expressed. These methylation patterns can be altered by treatment of the cells with 5-azacytidine or 5-azadeoxycytidine, but without a resultant activation of the type I procollagen genes. These results show that demethylation alone is not sufficient for gene activation, but that other signals are also required.

A segment of the type II procollagen gene has been isolated by screening a lambda Charon 4A library containing fragments of chicken genomic DNA. The specific clone, LgCOL(II), was selected by hybridization using overlapping inserts from two cDNA clones which are specific for a cartilage procollagen (Vuorio, E., Sandell, L., Kravis, D., Sheffield, V. C., Vuorio, T., Dorfman, A., and Upholt, W. B. (1982) Nucleic Acids Res. 10, 1175-1192). DNA sequence analysis of LgCOL(II) in the COOH-telopeptide region of the protein, shows conclusively that this DNA corresponds to the chicken type II procollagen gene. Hybridization of cDNA probes to restriction fragment gel blots together with DNA sequence analysis have established the orientation and position of the procollagen gene within the lambda Charon 4A vector and indicate that LgCOL(II) contains approximately 6 kilobase pairs of the type II procollagen gene plus additional DNA flanking the 3' end of the gene. DNA sequence analysis shows directly that LgCOL(II) contains DNA sequences identical with those in the cDNA clones. The portion of the gene from amino acid 578 of the triple helical region to the COOH-terminal end of the protein (approximately 700 amino acids) is contained within the clone, corresponding to approximately 50% of the amino acid coding sequence of the gene. This region of the chicken alpha 1 (type II) procollagen gene is encoded within a shorter segment of the chicken genome than is the corresponding region of the alpha 2(type I) procollagen gene.

Over 20 kilobase pairs of the human pro alpha 2(I) collagen gene have been isolated and characterized by restriction endonuclease mapping, cell-free translation of hybrid-selected RNA, and DNA sequence analysis. We have sequenced an exon and determined its length to be 108 base pairs (bp). This is consistent with the organization of chick and sheep collagen genes in that exons are multiples of 9 bp in length, frequently being 54 and 108 bp. The sequenced exon was bordered by a GT (guanine-thymine) at its 3' end and an AT (adenine-thymine) at its 5' end. This pattern has been found at all normal intron-exon junctions in eukaryotic cells. The amino acid sequence derived from DNA sequencing of this 108 bp exon revealed 88% homology compared to the amino acid sequence of bovine pro alpha 2(I). The bases encoded 12 Gly-X-Y triplets characteristic of the helical portion of collagen. A unique sequence Gly-Gly-Lys-Gly-Glu-Lys identified this fragment as alpha 2(I) collagen.

In a series of immortalized human bronchial epithelial cell lines continuing various oncogenes, the transcriptional levels of the type IV collagenase and the type IV procollagen genes were compared with the properties of invasiveness in vitro and tumorigenicity and metastatic ability in athymic nude mice. v-Ha-ras greatly enhanced invasion and metastasis, whereas v-Ki-ras, c-myc, and c-raf had lesser effects on these malignant phenotypes. In addition, cell lines derived from tumors obtained by injecting the original immortalized human bronchial epithelial cell lines into nude mice exhibited enhanced invasive and metastatic abilities and increased level of type IV collagenase mRNA when compared with the original immortalized human bronchial epithelial cell lines. Invasiveness and metastatic capacity correlated positively with expression of the type IV collagenase gene and negatively with the expression of the type IV procollagen gene, suggesting that these phenotypes are associated both with decreased production and increased dissolution of extracellular matrix.

Transformation of human lung fibroblasts (WI-38) by Simian Virus 40 (SV40) resulted in a decline of 25-30% in the amount of secreted collagen. The collagen produced by the transformed fibroblasts contained no type I collagen (i.e. alpha 1(I) and alpha 2 chains), which was the major collagen component produced by untransformed fibroblasts. Measurement of the procollagen mRNA levels by dot hybridization with nick-translated procollagen-cDNA clones showed that the absence of type I collagen was due to the absence of alpha 1(I) and alpha 2 procollagen mRNAs. This result was confirmed by hybridization of cDNA to total RNA with southern blots of the procollagen clones. To clarify the mechanism by which type I procollagen gene transcription is abolished in transformed cells, the methylation patterns of the alpha 1(I) and alpha 2 procollagen genes in normal and SV40-transformed fibroblasts were compared, using the chicken alpha 1(I) and alpha 2 procollagen-cDNA clones as probes. Methylated sites were detected by means of the restriction endonuclease isoschizomers HpaII and MspI. Methylation of the procollagen alpha 1(I) and alpha 2 genes was increased in the SV40-transformed fibroblasts, concurrently with the loss of type I collagen synthesis. DNA methylation may thus contribute to altered regulation of gene expression upon cell transformation.

Sixty kilobases of cloned DNA containing the entire human pro-alpha 2(I) collagen gene and 22 kilobases of flanking sequences have been isolated. Like the homologous avian gene, the 1366 amino acid residues of the human pre-pro-alpha 2(I) chain are encoded by 52 exons, whose relative locations and sizes have been determined. Analysis of the 5'- and 3'-untranslated regions have confirmed their exact lengths, as well as conclusively established the nature of five polymorphic mRNA transcripts.

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.

Type I collagen mutations in a group of patients with lethal perinatal osteogenesis imperfecta were identified in fibroblast RNA by a new method which can detect, by chemical modification and cleavage, single mismatched bases in heteroduplexes formed between mRNA and normal cDNA probes. Control cDNA probes spanning the area of the pro-alpha 1(I) and pro-alpha 2(I) chains likely to contain the mutations were radioactively labeled and used to form heteroduplexes with total patient RNA. Treatment of these heteroduplexes with hydroxylamine followed by cleavage of the cDNA strand at reactive bases by piperidine identified mismatches in the pro-alpha 1(I) cDNA in four patients. In the fifth patient a mismatch was detected in the pro-alpha 2(I) cDNA. To characterize these mutations the regions containing the mismatches were amplified by the polymerase chain reaction, cloned, and sequenced. All were heterozygous single base mutations which led to the substitution of glycine residues in the helical region of the pro-alpha-chains. The substitutions were pro-alpha 1(I) Gly973 and Gly1006 to Val, Gly928 to Ala, Gly976 to Arg, and pro-alpha 2(I) Gly865 to Ser. These mutations emphasize the importance of the Gly-X-Y repeating amino acid sequence for normal collagen helix formation and function in the extracellular matrix.

Although the major types of vertebrate collagen have a number of structural properties in common, significant DNA sequence homologies have not been detected between different portions of the helical coding domains within the same gene or between different genes. However, under non-stringen hybridization conditions we found considerable cross-homology within and between alpha 1(I) and alpha 2(I) chick cDNAs in the coding regions for helical sequences. Detailed analyses at the DNA sequence level have led us to propose that the gene for chick pro alpha 2(I) collagen arose from a 9-bp primordial sequence. A consensus sequence for the 9-bp repeat was derived: GGTCCTCCT, which codes for a Gly-Pro-Pro triplet. The primordial ancestor of this 9-bp unit, GGTCCTXCT, apparently underwent duplication and divergence. Each resulting 9-bp sequence was triplicated to form a 27-bp domain, and a condensation event produced a 54-bp domain. This genetic unit then underwent multiple rounds of amplification to form the ancestral gene for the full-length helical section of alpha 2(I). A different 9-bp consensus sequence (GGTCCCCCC) seems to have been the basis of the chick pro alpha 1(I) gene.

Structural defects in the human type 1 collagen genes are known to be the cause of several inherited disorders of connective tissue, such as osteogenesis imperfecta. The analysis and prenatal diagnosis of these disorders would be facilitated by establishing a set of polymorphic markers at these gene loci. We have previously reported the presence of an Msp 1 restriction fragment length polymorphism in the pro alpha 2 (1) collagen genes of several Southern African populations (Grobler-Rabie et al., in press). This report describes the detection of a Bgl II and an EcoRI polymorphism in the pro alpha 2 gene of South African Blacks.

Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.

We report the first isolation and identification of a mouse genomic fragment encoding amino acid sequences for the pro alpha 1(I) chain of type I procollagen. The DNA sequence of eight coding sequences is presented; five of these are 54 bp and three are 108 bp in length. Together these specify 198 amino acids which are 94% homologous to the corresponding bovine pro alpha 1(I) chain protein sequences. Each of the eight coding sequences is flanked by appropriate splice-junction sequences that exhibit considerable sequence complementarity to the rat small nuclear U1a RNA. In the 198 codons examined in this mouse genomic clone, the preferred codons for glycine and alanine are GGU (46/67) and GCU (23/30), respectively. This is in contrast to the codon usage reported for the chicken pro alpha 1(I) cDNA clone (Fuller and Boedtker, 1981). The examined coding sequences exhibit considerable nucleotide homology in both end-to-end and in staggered alignments. Based on an analysis of this homology data, a model is presented for the generation of 108-bp coding sequences from 54-bp units by two successive homologous recombinational events within coding sequences. Alternatively, the 108-bp units may have arisen by precise deletions of an intervening sequence between 54-bp coding sequences. Evidence supporting this is provided by a comparison of pro alpha 1(I) and pro alpha 2(I) genes. In the mouse pro alpha 1(I) gene amino acids 856-891 are encoded in a 108-bp unit; the chicken pro alpha 2(I) gene these residues are encoded in two 54-bp coding sequences. In addition, the coding sequences for nearly 50% of the alpha domain are condensed in the pro alpha 1(I) gene into a region approximately one half the size occupied by the comparable sequences in the pro alpha 2(I) gene.

Several cDNA clones for the human type II procollagen mRNA were isolated from a cartilage cDNA library. Six of the clones containing the longest inserts were subjected to restriction site mapping for alignment. All these six clones extended to the poly A tail. The longest clone, containing a 1470 bp insert, was named pHCAR3. Sequencing of pHCAR3 made it clear that neither of the two canonical AATAAA sequences of the human type II collagen gene is used as the polyadenylation signal. Two 60 bp stretches of high interspecies homology terminating in a hexanucleotide ATTAAA, located 23 nucleotides upstream of the poly A tail, apparently have an important role in determining the single polyadenylation signal for this gene. S1 protection experiments confirmed these observations.

HpCl, a Charon 4A bacteriophage containing a 16.3-kilobase insert of human genomic DNA, has been identified as representing the 3' portion of the human alpha 2(I) collagen gene by hybrid selected translation and cross-reactivity with characterized sheep and chick collagen genes. Evaluation of HpCl by restriction endonuclease mapping and alpha 2(I) mRNA hybridization demonstrated that the alpha 2(I) mRNA coding regions are separated by noncoding regions including one of 2.7 kilobase pairs near the 3' end of the mRNA coding region. In addition, a 1.8-kilobase XbaI-BamHI fragment containing the 2' a 1.8-kilobase XbaI-BamHI fragment containing the 3' end of the alpha 2(I) mRNA coding region includes a region of DNA that is repeated many times throughout the human genome. Quantification of alpha 2(I) gene number, using the technique of DNA dot hybridization, with two EcoRI fragments from HpClo as probes, demonstrated that HpCl is represented once in the human genome. In this context, it is unlikely that human alpha 2(I) collagen chain production is modulated by differential transcription of multiple alpha 2(I) genes.

We have isolated a genomic clone containing the 5'-terminal portion of the human pro-alpha 1(II)-collagen gene. This clone, HC2C, contains 10 kb of the gene and 6 kb of 5'-flanking sequences. It was selected by cross hybridization using a [32P]DNA probe containing the promoter region of the rat alpha 1(II)-collagen gene. We have determined the nucleotide sequence of the first exon and of 400 bp upstream. There is considerable homology between this human sequence and the corresponding rat sequence. As in the rat, the first exon contains a 155-bp untranslated segment and a 85-bp sequence coding for the signal peptide and a part of the N-propeptide of type-II procollagen. The segment preceding the transcription initiation site contains a conserved 'ATATAA' element, and several similar G + C-rich stretches. As in the rat gene, no 'CAT' element is evident between -70 and -120. We also find the sequence 5'-GTGGTCAGA-3' reported as an enhancer element in both viral and cellular genes, located around -290 bp. A high degree of homology exists between the atypical rat and human promoter structures; however, such homology is absent among the alpha 1(I), alpha 2(I) and alpha 1(III) promoters, which suggests that the unique alpha 1(II) sequences may be related to the specific expression of the alpha 1(II)-collagen gene.