The type X collagen gene. Intron sequences split the 5'-untranslated region and separate the coding regions for the non-collagenous amino-terminal and triple-helical domains

Type X collagen, expressed by hypertrophic chondrocytes, consists of homotrimeric molecules with subunits that are only about one-half the size of the polypeptides of fibrillar collagens. In this report we describe for the first time the complete primary structure of type X collagen, based on cloning and sequencing of cDNA and genomic DNA. A comparison between the nucleotide sequences of the cDNA and genomic DNA clones has also allowed determination of the complete exon structure of the type X collagen gene. Our results demonstrate that the primary translation product of the chicken type X collagen mRNA is 682 amino acid residues long with a calculated molecular mass of 67,317 Da for the nonhydroxylated form. This calculated molecular mass is in excellent agreement with the observed electrophoretic mobility of cell-free translation products with both poly(A)+ RNA isolated from chondrocytes as well as RNA transcribed in vitro from a full length cDNA construct. It is also in agreement with the observed size of type X collagen polypeptides isolated from the media of cultured hypertrophic chondrocytes. Thus, our data exclude the possibility of a high molecular weight precursor form of type X collagen. Our results also confirm that the chicken type X gene has a most unusual exon structure when compared to other vertebrate collagen genes. The gene has only three exons. One exon (97 base pairs (bp)), codes for most of the 5'-untranslated region of the mRNA, a second exon (159 bp) codes for the signal peptide and a short non-triple-helical domain, while the third exon (2136 bp) contains the coding region for the entire triple-helix and a large non-triple-helical carboxyl domain.     

Two-stage hormonal control of type IV collagen mRNA levels during differentiation of F9 teratocarcinoma cells

A cDNA clone related to mouse Type IV collagen has been prepared from F9 teratocarcinoma cells induced to differentiate with retinoic acid and dibutyryl-cAMP. This cDNA clone has been used to investigate the regulation of Type IV collagen mRNA during differentiation. The level of this mRNA is very low in untreated F9 cells, increases substantially after treatment of the cells with retinoic acid, and is further increased by addition of dibutyryl-cAMP. In contrast, dibutyryl-cAMP has no effect on the mRNA level in cells that have not been previously exposed to retinoic acid. These results demonstrate that these two compounds regulate in a sequential manner the steady-state level of Type IV collagen mRNA. This cDNA clone should allow a detailed examination of the mechanism of the two-stage regulation of collagen expression by retinoids and cyclic AMP.     

Cloning and sequencing of a Porifera partial cDNA coding for a short-chain collagen

Collagen is present in Porifera, the lowest multicellular animals, but there is no information available on the primary structure of the collagen chains in this phylum. Developing fresh-water sponges have been used to extract total RNA in order to study in vitro translation products and to construct a cDNA library. Four translated proteins were collagenase-sensitive (200 kDa, 160 kDa, 81 kDa and 48 kDa). The cDNA library was screened with a human collagen probe and a clone, EmC4, covering 1.2 kb was isolated. Nucleotide sequencing of EmC4 revealed a conceptual open reading frame coding for 366 amino acids terminated by a stop codon TGA with 103 nucleotides downstream. The presumed translation product encoded contained several domains: a non-collagenous C-terminal domain of 156 amino acids with 9 cysteines, an uninterrupted collagenous domain of 171 amino acids, a non-collagenous domain of 16 amino acids with 3 cysteines and a probably incomplete N-terminal collagenous domain of 23 amino acids. Comparison with other sequences suggested that this collagen chain might belong to a non-fibrillar collagen family which evolved into several sub-families giving rise to nematode cuticular collagens, and type IV collagens.     

The major progesterone-modulated proteins secreted into the sheep uterus are members of the serpin superfamily of serine protease inhibitors

The uterine milk proteins (UTMP) are a pair of structurally related basic glycoproteins that when newly synthesized carry phosphorylated mannosyl residues on their carbohydrate chains. They are the major proteins secreted by ovine endometrium under the influence of progesterone. RNA from a late pregnant ewe endometrium was isolated for use in in vitro translation assays and for constructing cDNA libraries. Translation experiments, initially with total cellular RNA and subsequently with RNA selected by hybridization with a specific cDNA, demonstrated the production of two polypeptides (Mr = 47,000 and 55,000) that were precipitated with antiserum to the UTMP. With microsomal membranes in the translation assay, there was increased production of an Mr = 57,000 form that was protected from protease digestion. Antibody screening of a cDNA library in lambda gt11 identified a short clone representing the 3' terminus of the mRNA that was shown by epitope selection experiments to be UTMP specific. This clone was then used to screen a lambda gt10 library. A longer clone (1.3 kilobases) was isolated and sequenced but lacked the 5' terminus to the mRNA. The latter sequence was obtained directly from the mRNA. Interesting features of the UTMP mRNA sequence, which was 1,352 bases long and contained a 1,287-base open reading frame, were two strong start codons, two potential sites for N-glycosylation and a repeat of 21 bases, six bases apart, that resulted in a repeat of seven amino acids. The inferred amino acid sequence agreed closely with the NH2-terminal amino acid sequence obtained directly from the UTMP and clearly placed the UTMP in the serpin superfamily of protease inhibitors. However, we have been unable to demonstrate inhibitory activity toward any serine protease so far tested.     

Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels.

Poly A containing RNA isolated from embryonic chick calvaria was transferred from 6% formaldehyde 0.75% agarose gels to diazobenzyloxymethyl paper and the paper then hybridized to either nick translated pro alpha 1 collagen cDNA clones, pCg1 or pCg54, or to the nick translated pro alpha 2 collagen cDNA clone, pCg45. From the mobilities of the bands hybridizing most strongly to each, pro alpha 2 collagen mRNA was shown to be slightly larger than pro alpha 1 mRNA; they are 5100 and 4900 nucleotides long respectively. pCg54 also hybridized weakly to two bands of lower mobility, corresponding to RNAs 6.4 and 5.6 kb long. Neither pCg54 nor pCg45 hybridized to type II procollagen mRNA in poly A containing RNA isolated from embryonic chick sterna.     

Isolation of cDNA and genomic DNA clones encoding type II collagen.

A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs.     

In vivo and in vitro studies of phage T4 lysozyme mRNA translation]

Translation of phage T4 lysozyme mRNA is studied in vivo and in vitro. Polyribosomes, carrying growing lysozyme polypeptides, are found to be homogenous enough and to contain 6 ribosomes. Complete molecules of phage lysozyme, which possess an enzymatic activity and are similar to the native enzyme in its electrophoretic mobility in polyacrylamide gel, have been synthetized in vitro on RNA isolated from phage-infected cells. The efficiency of RNA translation in cell-free system is discussed on the model of synthesis of functionally active individual protein.     

Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues

We have obtained cDNA clones coding for the A, B1, and B2 chains of laminin by screening a cDNA library prepared from mouse EHS tumor poly(A)RNA in the lambda gt11 expression vector with polyclonal antibody against denatured laminin. These cDNA clones were used in combination with a cDNA clone coding for the alpha 1 type IV collagen chain to study the regulation of genes for these basement membrane proteins in retinoic acid-induced differentiating mouse F9 teratocarcinoma cells and in various adult murine tissues. The levels of mRNA for the laminin A, B1, and B2 chains and for the alpha 1 type IV collagen chain were increased simultaneously and reached a maximum at almost the same time during the differentiation of F9 cells, suggesting coordinate expression in these cells. The tissue levels of mRNA encoding for the basement membrane components, however, varied considerably. The highest level of the B1 chain mRNA was observed in kidney, whereas, the levels of mRNA for A and B2 chains were highest in heart. Almost the same levels of expression of the alpha 1(IV) collagen mRNA were found in kidney, lung, and heart. The results indicate that the expression of genes for the basement membrane proteins is not coordinately regulated in these tissues. It is thus possible that different subunit structures of the laminin molecule may exist in tissues.     

Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.     

Isolation of cDNA clones for basal lamina components: type IV procollagen

We have isolated cDNA clones for mouse type IV procollagen from a library constructed from total poly A+RNA of 13.5 day mouse embryo parietal endoderm (PE) cells. In Northern analysis these clones hybridise to a 6.8 kb RNA which is abundant in embryonic PE cells and in differentiated F9 teratocarcinoma cells. Hybrid selection and in vitro translation of the cDNA specific mRNA produced a single polypeptide of Mr = 165 000. This polypeptide was specifically immunoprecipitated with mouse type IV procollagen antisera and comigrated on SDS-gel electrophoresis with one of the two in vitro synthesised chains of type IV procollagen. Undifferentiated F9 teratocarcinoma cells can be induced by retinoic acid and dibutyryl cAMP to differentiate in vitro into endoderm-like cells which resemble mouse PE cells in synthesising large amounts of basement membrane proteins, including type IV procollagen. Here we show, using one of the cDNA clones as a probe for type IV procollagen, that an increase in cellular concentration of type IV procollagen mRNA occurs within 24 to 48 hours of induction, reaching a constant high level by 72 hours.     

Isolation of an alpha 1 type-IV collagen cDNA clone using a synthetic oligodeoxynucleotide

We have isolated a cDNA clone (pCIV-1-225) for the alpha 1 subunit of basement membrane (type IV) collagen from a cDNA library made from Engelbreth-Holm-Swarm mouse tumor RNA. The cDNA library was screened with synthetic oligodeoxynucleotides derived from published amino acid (aa) sequences (Schuppan et al., 1982). Nucleotide sequence data established the identity of our cDNA clone to encode an alpha 1 type IV collagen. This clone contains 270 aa of the helical region and has three interruptions in the Gly-X-Y repeat unit.     

Processing Pisum sativum seed storage protein precursors in vitro

The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea(Pisum sativum) is complex,resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin,the major storage globulins.Translation in vitro of mRNAs hybridselected from mid-maturation pea seed RNAs by defined vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed;the derived polypeptides were of comparable sizes to those observed in vivo.The feasibility of transcribing mRNA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone,providing a method for determining polypeptide products of an expressed sequence.This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.     

Translation "in vitro" of globin mRNA from Xenopus laevis

RNA isolated from Xenopus laevis reticulocytes and characterized as globin mRNA (Meza et al., 1978) was tested for its capacity to stimulate "in vitro" a wheat germ translation system, and the ability to synthesize a polypeptide. The latter was identified as globin by its electrophoretic mobility and immunoprecipitation with antiglobin antibody.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.