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D P Bourque

Publications and source records attributed to D P Bourque.

16 recordsLinked to original sources
PubMed1992

Nuclear-encoded tobacco chloroplast ribosomal protein L24. Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis.

Using a Nicotiana tabacum leaf cDNA library in the expression vector lambda gt11, two cDNAs encoding the full-length precursor polypeptide (M(r) 20,696) of tobacco chloroplast ribosomal protein L24 were identified and sequenced. These cDNAs encode a mature protein of 146 amino acids (M(r) 16,418) with a transit peptide of 41 amino acids (M(r) 4,278). The mature tobacco L24 protein has 78, 65, 45, and 35% sequence identity with ribosomal proteins L24 of pea, spinach, Bacillus subtilis, and Escherichia coli, respectively. The transit peptide of tobacco L24 is 54 and 57% identical with that of L24 chloroplast ribosomal proteins of pea and spinach, respectively. An expressed beta-galactosidase:L24 fusion protein, bound to nitrocellulose filters, was used as affinity matrix to purify monospecific antibody to L24 protein. Using this monospecific antibody protein L24 was identified among high performance liquid chromatography (HPLC)-purified tobacco chloroplast ribosome 50 S subunit proteins. The predicted amino terminus of the mature L24 protein was confirmed by partial sequencing of the HPLC-purified L24 protein. Northern blot analysis revealed a single mRNA band (0.85-0.90 kilobase) corresponding in size to full-length L24 cDNA. The presence of multiple genes for L24 is suggested by Southern blot hybridization and characterization of two cDNAs for L24 which only differ in their 3'-noncoding sequences.

Amino Acid Sequence↗
PubMed1992

Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes.

A tobacco (Nicotiana tabacum cv. Petite Havana) leaf cDNA library was constructed in the expression vector lambda gt11. Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an M(r) 19,641 precursor to an M(r) 14,420 mature protein which is homologous to Escherichia coli ribosomal protein L27. One cDNA (L27-1; 882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly(A)29 tail. A beta-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins. Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins. The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27 protein. The mature L27 protein has 66%, 61%, 56%, and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E. coli, Bacillus stearo-thermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions. The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence. Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corresponding portion of yeast MRP7.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.

Amino Acid Sequence↗
PubMed1991

cDNA and deduced amino acid sequences of a dog liver cytochrome P-450 of the IIIA gene subfamily.

A 1.96 kbp cDNA encoding a male Beagle dog liver cytochrome P-450 of 503 amino acid residues (Mr 57,636) has been isolated and sequenced. The deduced amino acid sequence is 79.8%, 69.3% and 74.1% identical to the P450IIIA forms human NF25, rat PCN1 and rabbit LM3c, respectively. The amino terminal sequence is identical to the first 28 residues of the dog P450IIIA form PBD-1. Southern blot analysis yields restriction patterns consistent with IIIA gene subfamily multiplicity.

Amino Acid Sequence↗
PubMed1990

cDNA and deduced amino acid sequences of a dog hepatic cytochrome P450IIB responsible for the metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl.

The nucleotide sequence of a cDNA that codes for the major phenobarbital (PB)-inducible male beagle dog hepatic cytochrome P450 has been determined. Using a rabbit P450IIB cDNA probe (R. Gasser, M. Negishi, and R. M. Philpot, 1988, Mol. Pharmacol, 32, 22-30), a cDNA clone with a 2.6-kilobase pair insert was isolated from a lambda gt11 library prepared from hepatic mRNA from a PB-treated dog. The cloned insert was sequenced and found to contain an open reading frame coding for a polypeptide of 494 amino acids (Mr 56,183). The encoded protein can be assigned to the P450IIB subfamily on the basis of homology to cytochromes P450 from other species. The deduced amino acid sequence is 79% identical to that reported for rabbit P450 BO (P450IIB4) and 75% identical to that for rat P450b (P450IIB1). The sequence identity decreases to less than 52% when the dog sequence is compared with other P450II subfamilies. The deduced NH2-terminal 30 amino acids encoded by the dog cDNA are identical to those determined by sequence analysis of purified dog cytochrome P450 PBD-2, and the amino acid composition concurs with that determined for the PBD-2 protein (D. B. Duignan, I. G. Sipes, T. B. Leonard, and J. R. Halpert, 1987, Arch. Biochem. Biophys. 255, 290-303). Northern blots revealed two mRNA species of approximately 1.9 and 2.9 kilobases in length, which hybridized to the coding region of the dog P450IIB cDNA. The level of total hybridizable mRNA was increased approximately sixfold in livers from PB-treated dogs compared with that in untreated animals. This increase correlates well with the reported nearly sixfold increase in the level of PBD-2 protein and the fivefold increase in the rate of hepatic metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl following PB treatment. The two mRNA species may result from the use of different polyadenylation signals located in the 3'-noncoding region or from transcription of more than one gene for PBD-2. Southern blot analysis indicated that the dog P450IIB subfamily contains at least two closely related genes.

Amino Acid Sequence↗
PubMed1989

Translation of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.

Base Composition↗
PubMed1985

Derivation of a physical map of chloroplast DNA from Nicotiana tabacum by two-dimensional gel and computer-aided restriction analysis.

A combined approach was used to derive a detailed physical map of Nicotiana tabacum chloroplast DNA for the restriction enzymes SalI, SmaI, KpnI, and BamHI. Complete maps for the restriction enzymes SalI, SmaI, and KpnI were derived by using two-dimensional agarose gel analysis of fragments obtained by reciprocal double digestion of chloroplast DNA. We have characterized a complete cloned library of N. tabacum chloroplast DNA which contains overlapping restriction fragments resulting from partial digestion by BamHI. With these clones and existing data, we used a novel computer-aided analysis to derive a detailed map for the enzyme BamHI. A comparison and compilation of all published N. tabacum chloroplast DNA restriction maps is presented. Differences between ours and a previously published SmaI and BamHI restriction map are discussed.

Chloroplasts↗