Procollagen complementary DNA, a probe for messenger RNA purification and the number of type I collagen genes

Frischauf, Anna Maria; Lehrach, Hans; Rosner, Cynthia; Boedtker, Helga

doi:10.1021/bi00609a011

Cited by 37 publications

(19 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As an initial contribution to such an analysis we have isolated a highly purified mixture of the two type I procollagen mRNAs (2) and have prepared complementary DNA sequences to serve as hybridization probes for procollagen mRNA and gene sequences (3). Many experiments, however, require a far larger amount of highly purified procollagen sequences than can be obtained by mRNA isolation.…”

mentioning

confidence: 99%

Construction and characterization of a 2.5-kilobase procollagen clone.

Lehrach¹,

Frischauf²,

Hanahan³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

106

View full text Add to dashboard Cite

Recombinant bacterial plasmids have been constructed by inserting double-stranded chicken procollagen cDNA sequences linked to chemically synthesized decanucleotides containing HindIll sites into the HindIII site of pBR322. After transformation of Escherichia coli X1776, colonies were selected by ampicillin resistance and recombinants containing procollagen sequences were identified by colony hybridization to 32P-labeled procollagen cDNA. The inserts from three recombinant plasmids, pCglO, pCgl3, and pCg45, were 1200, 2200, and 2550 base pairs long, respectively. Their sequence homolo has been established by restriction mapping and crosshybri ization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts. pCg45 has been positively identified as containing the pro a2 collagen sequence by partial determination of the DNA sequence of its ends: it has a short thymine-rich sequence at one end and a sequence coding for residues 478-499 in the chicken a2 chain at the other end.The analysis of the regulatory mechanisms that ensure the tightly controlled tissue and developmental stage-specific expression of the different collagen genes (1) will be essential for the understanding of developmental processes in eukaryotes. As an initial contribution to such an analysis we have isolated a highly purified mixture of the two type I procollagen mRNAs (2) and have prepared complementary DNA sequences to serve as hybridization probes for procollagen mRNA and gene sequences (3). Many experiments, however, require a far larger amount of highly purified procollagen sequences than can be obtained by mRNA isolation. To obviate this difficulty, we have used recombinant DNA technology to amplify procollagen cDNA sequences in Escherichia coli. Clones containing procollagen cDNA sequences were obtained using modifications of the method used by Goodman and his colleagues to clone rat proinsulin cDNA (4). We present here a description of three pro a2 clones characterized thus far. MATERIALS AND METHODSConstruction of Recombinant Plasmids. Procollagen mRNAs, purified by binding embryonic chicken calvaria poly(A)-containing RNA to Sepharose 4B as described (3), served as template for the synthesis of the cDNA used for cloning. Double-stranded cDNA was synthesized and ligated to chemically synthesized decanucleotides containing HindIll sites (unpublished data). In brief, double-stranded cDNA was prepared by a procedure in which single-stranded cDNA is not isolated prior to second strand synthesis (5). It was converted into perfect duplex molecules with blunt ends by digestion with single-strand nuclease S1 from Aspergillus oryzae [purified from amylase (Sigma) as described by Vogt (6) as follows: 200-ml cultures of X1776 were grown to an optical density at 600 nm of 0.2/ml, centrifuged down, resuspended in 20 ml of 10 mM NaCl, repelleted, and resuspended in 10 ml of 70 mM MnCl2/40 mM Na acetate, pH 5.6/30 mM CaC12. A 0.2-ml portion of the suspension was then added to sterile tubes containing 25 or 100 ng of...

show abstract

mentioning

confidence: 99%

Construction and characterization of a 2.5-kilobase procollagen clone.

Lehrach¹,

Frischauf²,

Hanahan³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

106

View full text Add to dashboard Cite

show abstract

“…Instead, band B was more heavily labelled than band A, which was often so faint as to be undetectable except with marked over-exposure of the fluorogram. In this context, it is noteworthy that a close examination of fluorograms in some previous reports in which the wheat-germ system was used (Adams et al, 1977;Frischauf et al, 1978;Howard et al, 1978) also show a similar heavier labelling of the band corresponding Vol. 182 to pro-a2 chains as compared with pro-al chains.…”

Section: Discussionmentioning

confidence: 52%

“…We initially chose the wheat-germ cell-free system (Roberts. & Paterson, 1973) for its ease of preparation, low endogenous mRNA content and because it had been used successfully by others to translate procollagen mRNA (Harwood et al, 1975;Boedtker et al, 1976;Adams et al, 1977;Frischauf et al, 1978;Howard et al, 1978). Because wheat germ from different commercial sources yields cell-free systems of differing efficiencies, the wheat germ used in this study was obtained from the source reported by Marcu & Dudock (1974) to give extracts with the best cell-free synthesis.…”

Section: Resultsmentioning

confidence: 99%

“…Decreases in rye-embryo viability are also associated with the development of lesions in rRNA and increases in endogenous nuclease activity (B. E. Roberts, 1972;Osborne et al, 1974;Cheah & Osborne, 1978). All these factors could similarly contribute to the variability in translation efficiencies of cell-free systems derived from different wheatgerm stocks (Marcu & Dudock, 1974) and explain, at least in part, the complete translation of procollagen mRNA by some (Harwood et al, 1975;Boedtker et al, 1976;Adams et al, 1977;Frischauf et al, 1978;Howard et al, 1978) and the failure or incomplete success by others (Neufang et al, 1975;Benveniste et al, 1976;Zeichner & Rojkind, 1976). It is of note, however, that, even where successful translation of procollagen mRNA has been reported, in most of these studies the synthesis of pro-al and pro-a2 chains in a 2: 1 ratio was not achieved.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

Cheah

Grant

Jackson

1979

Biochemical Journal

View full text Add to dashboard Cite

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-a chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-a chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-al and pro-a2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2: 1 ratio of pro-al and pro-a2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-al and pro-a2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the 'Signal Hypothesis'.

show abstract

“…It has been found to be monocistronic [14] and its M, has been estimated to be 1.5 x lo6 for each of the three polypeptide chains composing the precursor of the collagen molecule. This large mRNA has been translated in different endogenous [15] or exogenous cellfree systems such as the wheat germ [4][5][6][7]111, the rabbit reticulocyte lysate [3,8,10,12] and the Krebs ascites tumor [9] to yield precursors of a1 and a2 chains.…”

mentioning

confidence: 99%

Extraction and Translation of Collagen mRNA from Fetal Calf Skin

Kaufmann

Belayew

Nusgens

et al. 1980

European Journal of Biochemistry

View full text Add to dashboard Cite

RNA was extracted from fetal calf skin by two different procedures, using phenol or guanidine hydrochloride. Poly(A)-rich RNA was separated by oligo(dT)-cellulose affinity chromatography and was further fractionated by sucrose density gradient centrifugation. When translated in an optimized wheat germ extract cell-free system, unfractionated guanidine-hydrochloride-extracted poly(A)-rich RNA directed the synthesis of two collagenase-sensitive protein bands, while phenolextracted poly(A)-rich RNA with a sedimentation coefficient higher than 25 S was the only fraction to direct the same synthesis. On the basis of their electrophoretic mobility on a sodium dodecylsulfate/ urea/polyacrylamide gel, these proteins were identified with procollagen stl(1) and procollagen a2. Inhibition of translation by phenol-extracted poly(A)-rich RNA with a sedimentation coefficient lower than 25 S was also observed.Guanidine-hydrochloride-extracted poly(A)-rich RNA from fetal calf skin directed the synthesis of three distinct collagenase-sensitive proteins in the micrococcal-nuclease-digested rabbit reticulocyte cell-free system ; these seemed to correspond to procollagen a1 (I), procollagen a2 and procollagen a1 (I1 I).Several different types of collagens are known to compose the resistant framework which ensures the physical properties of connective tissues (for review, see [l]). Collagen mRNA has been isolated from various connective tissues, for example, tendon [2,3], bone [4-71, lung [8] and fibroblast culture [3,9-131. It has been found to be monocistronic [14] and its M, has been estimated to be 1.5 x lo6 for each of the three polypeptide chains composing the precursor of the collagen molecule. This large mRNA has been translated in different endogenous [15] or exogenous cellfree systems such as the wheat germ [4-7,111, the rabbit reticulocyte lysate [3,8,10,12] and the Krebs ascites tumor [9] to yield precursors of a1 and a2 chains.Skin is a much more complex tissue since it is composed of a parenchyme of ectodermal origin (the epidermis and the epidermal derived appendages) and a connective tissue. Its fibrous framework predominantly consists of types I and 111 collagen in measurable concentrations [16,17], as well as other types of collagens in trace amounts. Skin, therefore, represents a potential source of several mRNA species.Skin is a most interesting tissue for studying the genetic expression of the various collagen species since the proportion of type I11 to type I collagen undergoes changes during development, wound healing [16,17] and various pathological processes. In order to approach the control mechanism regulating such changes, suitable techniques have been devised for extraction and translation of skin mRNA. This paper compares the efficiency of the phenol method compared to the guanidine hydrochloride method for skin RNA extraction. The isolated RNAs, purified by oligo(dT)-cellulose affinity chromatography and sucrose gradient ultracentrifugation, were translated in two different cell-free systems. The pres...

show abstract

Procollagen complementary DNA, a probe for messenger RNA purification and the number of type I collagen genes

Cited by 37 publications

References 23 publications

Construction and characterization of a 2.5-kilobase procollagen clone.

Construction and characterization of a 2.5-kilobase procollagen clone.

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

Extraction and Translation of Collagen mRNA from Fetal Calf Skin

Contact Info

Product

Resources

About