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H Boedtker

Publications and source records attributed to H Boedtker.

34 records · Page 2Linked to original sources
PubMed1981

Fine structural analysis of the chicken pro alpha 2 collagen gene.

Forty-two kilobase pairs of cloned chicken DNA containing 80% of the pro alpha 2 (type I) collagen gene and 8 kilobase pairs of 3' flanking sequences have been isolated. Detailed analysis of these clones indicates that this collagen gene spans approximately 40 kilobase pairs of DNA and contains on the order of 50 introns. The fine structure of 40% of the pro alpha 2 gene, including its 3' end, was determined by Southern blot restriction endonuclease mapping using a 2.6-kilobase pair procollagen cDNA clone pCg45, as a probe, and by DNA sequence determination of more than 2 kilobase pairs of this part of the genome. Exons in the triple-helical coding region are all multiples of the 9 base pairs coding for the Gly-X-Y triplet and vary in size from 45 to 108 base paris. The sequences of all six exons in a 3.8-kilobase pair EcoRI fragment were determined. One of these, a 249-base pair exon, joins the collagen domains; it codes for the last 15 amino acids of the triple-helical coding region, the telopeptide, and the first 53 amino acids of the carboxy-terminal propeptide.

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PubMed1979

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.

Animals↗
PubMed1978

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

Type I procollagen mRNAs were separated from contaminating low-abundance messenger and nuclear RNAs by chromatography over Sepharose 4B in 0.65 M NaCl at room temperature. All of 27S rRNA and four-fifths of procollagen mRNAs bind to Sepharose under these conditions, while 18S rRNA and about three-fourths of other poly(A)-containing RNAs do not bind. AMV reverse transcriptase was used to prepare complementary DNA to procollagen mRNA at each purification step. Hybridization studies, in RNA excess, were carried out to establish the enrichment at each step both with respect to total RNA and to poly(A)-containing RNA. While "purified" procollagen mRNA preparations still consist of about 50% 27S rRNA, over 80% of cDNA prepared from it back hybridizes to its template at a log of cr0t1/2 of -1.9. This type I procollagen cDNA hybridizes in DNA excess to DNA isolated from chicken erythrocytes and from embryonic chick calvaria at a log c0t1/2 of 3.1, demonstrating that procollagen cDNA is complementary to unique gene sequences in both tissues and that procollagen genes are not reiterated.

Animals↗
PubMed1978

Construction and characterization of a 2.5-kilobase procollagen clone.

Recombinant bacterial plasmids have been constructed by inserting double-stranded chicken procollagen cDNA sequences linked to chemically synthesized decanucleotides containing HindIII sites into the HindIII site of pBR322. After transformation of Escherichia coli chi1776, 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, pCg10, pCg13, and pCg45, were 1200, 2200, and 2550 base pairs long respectively. Their sequence homology has been established by restriction mapping and crosshybridization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts, pCg45 has been positively identified as containing the pro alpha2 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 alpha2 chain at the other end.

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PubMed1976

Isolation and translation of calvaria procollagen messenger ribonucleic acids.

Procollagen messenger ribonucleic acid (mRNA) was isolated from the calvaria of 15-day-old chick embryos by chromatographing total RNA over oligo(dT)-cellulose two times, and then fractionating the twice-bound RNA on 85% Me2SO/0-20% sucrose gradients. When analyzed on 99% formamide gels, the 27-30S fraction had three sharp fluorescent bands, one corresponding to 27S ribosomal RNA (rRNA), the others having mobilities lower than 27S corresponding to molecular weights of 1 700 000 and 1 800 000. In wheat-germ, cell-free extracts, the 27-30S fraction directed the synthesis of two prominent collagenase sensitive polypeptides with mobilities corresponding to the calvaria pro-alpha chain markers. Twelve percent of this [3H]proline-labeled, wheat-germ product could be hydroxylated with prolyl hydroxylase.

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PubMed1974

The identification of collagen messenger RNA.

RNA isolated from calvaria of 16- to 18- day-old chick embryos, assayed in rabbit reticulocyte lysates, programs the synthesis of a collagenase-sensitive protein with the molecular weight of collagen pro-alpha-chains. When RNA labeled with [(3)H]uridine for 2 hr and chased for 1 or 2 hr was electrophoresed on aqueous polyacrylamide gels, most of the radioactivity not in 28S or 18S rRNA migrated with an apparent molecular weight of about 1,800,000. After oligo(dT)-cellulose chromotography and analysis in 99% formamide gels, this nonribosomal, rapidly labeled calvaria RNA species migrates at 28S-30S and thus has a molecular weight of at least 1,600,000. Both the ability to program the synthesis of collagenase-sensitive protein in reticulocyte lysates and the presence of a single prominent rapidly labeled 30S peak in acrylamide gels strongly support the deduction that there is only one major mRNA species in calvaria and that this species is collagen messenger RNA.

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