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P L Davies

Publications and source records attributed to P L Davies.

9 recordsLinked to original sources
PubMed1979

Sequences of large T1 ribonuclease-resistant oligoribonucleotides from protamine mRNA: the overall architecture of protamine mRNA.

Limited T1 ribonuclease digestion of the family of protamine mRNA's purified from rainbow trout testis yields several large oligoribonucleotide fragments ranging in size from 12--54 nucleotides in length. Several of these fragments purified by two dimensional gel electrophoresis contain several G residues and must represent nuclease-resistant, base-paired regions of the mRNA. Sequence analysis of these oligonucleotides by the method of Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and Guilley, H. (1977) Nature 269: 833-836, shows that these oligoribonucleotides arise from the 5'- and 3'-non-coding regions of the mRNA. Comparisons of the sequences of the large RNA fragment with DNA sequences obtained after cloning double-stranded protamine cDNA in the plasmids pBr322 and pmB9 show precise correspondence of a 54 nucleotide RNA fragment with positions 49--100 from the 3'-poly(A) tract and extending to within 5 nucleotides of the termination codon. Two other RNA fragments of 21 and 25 nucleotides in length arise from the 5'-non-coding region of the message and possess an AUG-sequence at their 3'-termini which is the initiation codon. The presence of distinct by homologous sequences in several sets of large RNA fragments is consistent with the presence of several closely related protamine mRNA's.

Animals↗
PubMed1979

Double-stranded protamine cDNA: synthesis and characterization.

Double-stranded protamine complementary DNA (cDNA) was synthesized from a protamine mRNA template via the single-stranded cDNA intermediate using avian myeloblastosis virus reverse transcriptase. Synthesis at 37 and 46 degrees C resulted in similar overall yields (greater than or equal to 18%), although the initial rate of synthesis was higher at 46 degrees C than at 37 degrees C. The DNA of the second strand of the double-stranded cDNA product was 84% resistant to prolonged digestion with excess S1 nuclease. The S1 nuclease resistant material ranged in size from 235 to 100 base pairs (bp) with an average length of 185 bp. Analysis of the products released from double-stranded protamine cDNA by depurination indicated that there were a number of cytosine-rich oligopyrimidine tracts in protamine mRNA, namely C4, C4U1, C5U1, C6U1, C6U4, and C7U1. On the basis of the amino acid sequences for rainbow trout protamines, C5U1, C6U1 and C7U1 must be located within the noncoding regions. Double-stranded protamine cDNA was cleaved at least once by the restriction endonucleases HaeIII and HhaI and in several places by HpaII. These restriction endonucleases cleave at sequences which have a high probability of occurring within the coding region of protamine mRNA, again based on the known amino acid sequences of the rainbow trout protamines.

DNA↗
PubMed1979

A single-breath method of alveolar O2 determination.

We designed a single-breath method of alveolar O2 determination, requiring only a single tidal breath expirate and concomitant arterial blood sample. The PAO2 equation used in our method was derived by applying the Bohr equation to both O2 and CO2 and independant of VO2, VCO2 and RQ. In 35 patients with different degrees of airway obstruction, at rest and during exercise, the single-breath method agreed well with the classic method of PAO2 determination which required 3 min of expired gas collection and derivation of VO2, VCO2 and RQ. The mean difference between the PAO2 estimates by the two methods was 0.36 mm Hg as calculated for all patients, both at rest and during exercise. At rest, in 2 out of 35 cases the difference was greater than 2 mm Hg and such difference happened only in 1 case during exercise. A good correlation existed between the two PAO2 estimates by the two methods (r ranging from 0.977 to 0.996). The data indicated that single-breath method of PAO2 determination was reliable. Its extreme simplicity would facilitate greatly the assessment of gas exchange efficiency in situations where both patient's cooperation and laborator equipment are less than optimum.

Adult↗
PubMed1977

Protamine messenger RNA from rainbow trout testis contains the nucleotide sequence A-A-U-A-A-A in an untranslated region.

Full-length, complementary DNAs were prepared to rainbow trout protamine mRNA using reverse transcriptase and were labelled during synthesis by the replacement of dATP by [alpha32P]dATP or dTTP by [alpha32P]dTTP. The 32P-labelled protamine complementary DNAs were digested with T4 endonuclease IV. Fragments from the digests were separated in two dimensions, and those discrete fragments which could be identified from both the A-labelled and T-labelled complementary DNAs were subjected to sequence analysis. The sequences described here all arise from the non-coding region. One pentadecanucleotide contained the sequence A-A-U-A-A-A which has been reported by Proudfoot and Brownlee ((1976) Nature 263, 211-214) to occur in the noncoding regions of six other eukaryotic mRNAs.

Animals↗
PubMed1977

Identification and isolation of protamine messenger ribonucleoprotein particles from rainbow trout testis.

Treatment of rainbow trout testis polyribosomes with ethylenediaminetetraacetic acid released polyadenylated protamine messenger RNA in the form of a ribonucleoprotein (mRNP) particle. This mRNP particle which sedimented at 12-14 S could be identified by hybridization to [3H]poly(U) and was partially purified by two successive sucrose gradient sedimentations. When RNA was extracted from the mRNP particle and used as a template in the wheat germ cell-free protein synthesizing system the sole product of translation was protamine. Of this RNA, 30% contained poly(A) sequences and was shown to comigrate with polyadenylated protamine messenger RNA during polyacrylamide gel electrophoresis. When the proteins of the mRNP particle were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two prominent polypeptides with apparent molecular weights of 73 000 and 29 000 appeared reproducibly. Treatment of trout testis polyribosomes with puromycin in the presence of 0.5 M KCl released a smaller (8-10S) mRNP particle which was similarly shown to contain protamine messenger RNA. Trout testis postribosomal supernatant fraction possessed 16-18S mRNP particles containing polyadenylated RNA which cosedimented with protamine messenger RNA when the particles were dissociated with sodium dodecyl sulfate.

Animals↗
PubMed1977

Sequence analysis of protamine mRNA from the rainbow trout. Depurination and nearest neighbor analysis of protamine cDNA.

Protamine cDNA, which was a full length copy of protamine mRNA was labeled during its synthesis by using deoxynucleoside [alpha-32P]triphosphates. Depurination analysis showed that there were 19 different pyrimidine oligonucleotides in protamine cDNA, some of which contained isomeric sequences. The stoichiometry of the pyrimidine oligonucleotides indicated that, while some sequences probably occur in each of the protamine mRNA components, other sequences are clearly absent from one or more of the components. Several of the pyrimidine oligonucleotides had sequences consistent with the amino acid sequences of the rainbow trout protamines. The longest oligopyrimidine tract, C7T4, had a complementary RNA sequence of AGGAGAGGAGG, a stoichiometry of close to 1, and fitted the amino acid sequence Arg-Arg-Gly-Gly which occurs near the COOH terminus of each of three major protamine components. Other pyrimidine oligonucleotides analyzed were complementary to RNA sequences from the noncoding region of protamine mRNA. There appears to be no preferential use of one particular arginine codon or set of codons. Of the 21 to 22 arginine codons in protamine mRNA no less than 7 and no more than 12 are of the CGX series. The other two codons, AGA and AGG, both occur but not in a series of more than two together. This indicates that the RNA sequences coding for the arginine tracts tend to contain a mixture of arginine codons. Nearest neighbor frequency analysis of protamine cDNA gives a low value for the frequency of the CpG doublet, despite its occurrence in four out of the six arginine codons. This is in accordance with the observation that the sequence CpG is surprisingly rare in vertebrate DNA and in the RNA transcribed from it.

Animals↗