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Papers by patricia Ashley

Pediatric Research, 1987

ABSTRACT

Proceedings of The National Academy of Sciences, 1982

We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikre... more We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikrein. Four cloned overlapping double-stranded cDNAs gave a continuous mRNA sequence of 867 nucleotides beginning within the 5'-non-coding region and extending to the poly(A) tail. The mRNA sequence reveals that pancreatic kallikrein is synthesized as a prezymogen of 265 amino acids, including a proposed secretory prepeptide of 17 amino acids and a proposed activation peptide of 11 amino acids. The activation peptide, although similar in length, is distinct from those of the other classes of pancreatic serine proteases. The amino acid sequence of the predicted active form of the enzyme is closely related to the partial sequences obtained for other kallikrein-like serine proteases including rat submaxillary gland kallikrein, pig pancreatic and submaxillary gland kallikreins, the γ subunit of mouse nerve growth factor, and rat tonin. Key amino acid residues thought to be involved in the substrate-cleavage specificity of kallikreins are retained. Hybridization analysis showed relatively high levels of kallikrein mRNA in the rat pancreas, submaxillary and parotid glands, spleen, and kidney, indicating the active synthesis of kallikrein in these tissues.

Proceedings of The National Academy of Sciences, 1984

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts ≈ 425 and ≈ 950 base pairs long... more Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts ≈ 425 and ≈ 950 base pairs long, respectively, which are specific for bovine cholesterol sidechain cleavage cytochrome P-450 (P-450scc), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450scc with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450scc is encoded by mRNA species ≈ 2000 bases long, a majority of which are polyadenylylated. P-450scc mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450scc mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.

Biochemistry, 1985

Four distinct kallikrein-related mRNAs (PS, S1, S2, and S3), encoded by members of a multigene fa... more Four distinct kallikrein-related mRNAs (PS, S1, S2, and S3), encoded by members of a multigene family, are selectively expressed in various combinations in several rat tissues. Although closely related along most of the mRNA sequence, the four mRNAs can be selectively detected with synthetic oligonucleotide probes complementary to highly variable mRNA subregions. PS mRNA, which encodes an enzyme with true kallikrein activity, is present at high levels in the submaxillary gland, pancreas, and kidney. S1 mRNA, which encodes an enzyme similar to the PS kallikrein, is detected only in the submaxillary gland and is present at one-fifth the PS mRNA level. S2 mRNA, which encodes the enzyme tonin, is present in the submaxillary gland at half the PS mRNA level and at a slightly higher level in the prostate. S3 mRNA, which encodes an enzyme very similar to tonin, is present in the submaxillary gland at one-tenth the PS mRNA level and in the prostate at about the same level as tonin mRNA.

Biochemistry, 1985

W e have determined the nucleotide sequence of four submaxillary gland mRNAs, designated PS, S1, ... more W e have determined the nucleotide sequence of four submaxillary gland mRNAs, designated PS, S1, S2, and S3, that encode kallikrein and kallikrein-like serine proteases. The four enzymes share between 74% and 86% amino acid sequence identity and are identical in length with the exception of single two amino acid deletions in the S2 and S3 enzymes. The PS enzyme appears to be a true tissue kallikrein. The S 1 enzyme shares 86% amino acid sequence homology with the PS enzyme and retains key amino acid residues thought to be primary determinants of kallikrein cleavage specificity. The S 2 enzyme is rat submaxillary tonin. The amino acid sequence of the S3 enzyme is identical with tonin at 84% of its amino acid positions and retains the same amino acid substitutions at positions likely to determine substrate cleavage preferences.

Biochemical and Biophysical Research Communications, 1980

Biochemical and Biophysical Research Communications, 1987

Archives of Biochemistry and Biophysics, 1984

A radical species of monochlorodimedone has been characterized by its high reactivity with molecu... more A radical species of monochlorodimedone has been characterized by its high reactivity with molecular Oz. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous HzOz only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of Hz02 added and Oz consumed. However, with excess Br-and horseradish peroxidase, or high Br-or Cl-and chloroperoxidase, a 1:l stoichiometry between HzOz and the substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per HzOz. Moreover, in the absence of halide, at substrate:H202 ratios greater than 2.0, chloroperoxidase catalyzed significant Oz uptake; this enzymedependent autoxidation of monochlorodimedone also occurred in the presence of Clor Br-, when HzOz was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Cwrnmun. 116, 873-8791, strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOC1 formation catalyzed by chloroperoxidase are considered.

Archives of Biochemistry and Biophysics, 1981

Analytical Biochemistry, 1984

A convenient and rapid technique for preparing radiolabeled single-stranded DNA hybridization pro... more A convenient and rapid technique for preparing radiolabeled single-stranded DNA hybridization probes has been developed. Single-stranded recombinant M13 phage DNA containing the mRNA strand of a cloned cDNA is bound to diazobenzyloxymethyl-cellulose in a manner that permits the synthesis of a complementary DNA using reverse transcriptase and primed with either oligo(dT) or the M13 single-stranded primer. A procedural advantage is that after synthesis the unincorporated radiolabeled nucleotides are washed away easily, and the radiolabeled single-stranded DNA probe is eluted with formamide, ready for use. To limit the DNA copy to the insert, a preliminary synthesis reaction is performed with unlabeled nucleotides, primer, and enzyme, followed by digestion of the reaction mix with a restriction endonuclease that recognizes a unique site in the recombinant immediately upstream of the cDNA insert. After elution of the unlabeled synthesized complementary DNA, a second synthesis reaction yields highly radiolabeled single-stranded DNA that extends only the length of the mRNA insert. A major advantage is that the restriction enzyme-cleaved, cellulose-bound template can be stored and reused repeatedly.

Pediatric Research, 1987

ABSTRACT

Proceedings of The National Academy of Sciences, 1982

We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikre... more We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikrein. Four cloned overlapping double-stranded cDNAs gave a continuous mRNA sequence of 867 nucleotides beginning within the 5'-non-coding region and extending to the poly(A) tail. The mRNA sequence reveals that pancreatic kallikrein is synthesized as a prezymogen of 265 amino acids, including a proposed secretory prepeptide of 17 amino acids and a proposed activation peptide of 11 amino acids. The activation peptide, although similar in length, is distinct from those of the other classes of pancreatic serine proteases. The amino acid sequence of the predicted active form of the enzyme is closely related to the partial sequences obtained for other kallikrein-like serine proteases including rat submaxillary gland kallikrein, pig pancreatic and submaxillary gland kallikreins, the γ subunit of mouse nerve growth factor, and rat tonin. Key amino acid residues thought to be involved in the substrate-cleavage specificity of kallikreins are retained. Hybridization analysis showed relatively high levels of kallikrein mRNA in the rat pancreas, submaxillary and parotid glands, spleen, and kidney, indicating the active synthesis of kallikrein in these tissues.

Proceedings of The National Academy of Sciences, 1984

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts ≈ 425 and ≈ 950 base pairs long... more Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts ≈ 425 and ≈ 950 base pairs long, respectively, which are specific for bovine cholesterol sidechain cleavage cytochrome P-450 (P-450scc), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450scc with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450scc is encoded by mRNA species ≈ 2000 bases long, a majority of which are polyadenylylated. P-450scc mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450scc mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.

Biochemistry, 1985

Four distinct kallikrein-related mRNAs (PS, S1, S2, and S3), encoded by members of a multigene fa... more Four distinct kallikrein-related mRNAs (PS, S1, S2, and S3), encoded by members of a multigene family, are selectively expressed in various combinations in several rat tissues. Although closely related along most of the mRNA sequence, the four mRNAs can be selectively detected with synthetic oligonucleotide probes complementary to highly variable mRNA subregions. PS mRNA, which encodes an enzyme with true kallikrein activity, is present at high levels in the submaxillary gland, pancreas, and kidney. S1 mRNA, which encodes an enzyme similar to the PS kallikrein, is detected only in the submaxillary gland and is present at one-fifth the PS mRNA level. S2 mRNA, which encodes the enzyme tonin, is present in the submaxillary gland at half the PS mRNA level and at a slightly higher level in the prostate. S3 mRNA, which encodes an enzyme very similar to tonin, is present in the submaxillary gland at one-tenth the PS mRNA level and in the prostate at about the same level as tonin mRNA.

Biochemistry, 1985

W e have determined the nucleotide sequence of four submaxillary gland mRNAs, designated PS, S1, ... more W e have determined the nucleotide sequence of four submaxillary gland mRNAs, designated PS, S1, S2, and S3, that encode kallikrein and kallikrein-like serine proteases. The four enzymes share between 74% and 86% amino acid sequence identity and are identical in length with the exception of single two amino acid deletions in the S2 and S3 enzymes. The PS enzyme appears to be a true tissue kallikrein. The S 1 enzyme shares 86% amino acid sequence homology with the PS enzyme and retains key amino acid residues thought to be primary determinants of kallikrein cleavage specificity. The S 2 enzyme is rat submaxillary tonin. The amino acid sequence of the S3 enzyme is identical with tonin at 84% of its amino acid positions and retains the same amino acid substitutions at positions likely to determine substrate cleavage preferences.

Biochemical and Biophysical Research Communications, 1980

Biochemical and Biophysical Research Communications, 1987

Archives of Biochemistry and Biophysics, 1984

A radical species of monochlorodimedone has been characterized by its high reactivity with molecu... more A radical species of monochlorodimedone has been characterized by its high reactivity with molecular Oz. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous HzOz only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of Hz02 added and Oz consumed. However, with excess Br-and horseradish peroxidase, or high Br-or Cl-and chloroperoxidase, a 1:l stoichiometry between HzOz and the substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per HzOz. Moreover, in the absence of halide, at substrate:H202 ratios greater than 2.0, chloroperoxidase catalyzed significant Oz uptake; this enzymedependent autoxidation of monochlorodimedone also occurred in the presence of Clor Br-, when HzOz was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Cwrnmun. 116, 873-8791, strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOC1 formation catalyzed by chloroperoxidase are considered.

Archives of Biochemistry and Biophysics, 1981

Analytical Biochemistry, 1984

A convenient and rapid technique for preparing radiolabeled single-stranded DNA hybridization pro... more A convenient and rapid technique for preparing radiolabeled single-stranded DNA hybridization probes has been developed. Single-stranded recombinant M13 phage DNA containing the mRNA strand of a cloned cDNA is bound to diazobenzyloxymethyl-cellulose in a manner that permits the synthesis of a complementary DNA using reverse transcriptase and primed with either oligo(dT) or the M13 single-stranded primer. A procedural advantage is that after synthesis the unincorporated radiolabeled nucleotides are washed away easily, and the radiolabeled single-stranded DNA probe is eluted with formamide, ready for use. To limit the DNA copy to the insert, a preliminary synthesis reaction is performed with unlabeled nucleotides, primer, and enzyme, followed by digestion of the reaction mix with a restriction endonuclease that recognizes a unique site in the recombinant immediately upstream of the cDNA insert. After elution of the unlabeled synthesized complementary DNA, a second synthesis reaction yields highly radiolabeled single-stranded DNA that extends only the length of the mRNA insert. A major advantage is that the restriction enzyme-cleaved, cellulose-bound template can be stored and reused repeatedly.