Search PubMed⌕ Search

Biomedical subjects

R J MacDonald

Publications and source records attributed to R J MacDonald.

At least 55 records · Page 3Linked to original sources
PubMed1986

Computer aided prediction and evaluation of the tertiary structure for rat elastase II.

Predictive methods have been extended to generate a proposed tertiary structure of rat elastase II on the basis of primary amino acid sequence and structural homologies within the family of mammalian serine proteinases. Force field refinement calculations were used to relax the structure. Structurally conserved molecules of solvation were introduced and the structure was again refined by means of force-field calculations. The resulting structure suggests probable substrate cleavage preferences. An independent statistical analysis of the crystallographically refined structures of serine proteinases (0.01-0.13 A, RMS) shows a close similarity to the final predicted model of rat pancreatic elastase II (0.03-0.14 A, RMS).

Amino Acid Sequence↗
PubMed1986

Transgenic progeny inherit tissue-specific expression of rat elastase I genes.

Six different lines of transgenic mice bearing rat elastase I genes stably inherit both the high-level pancreatic expression and low-level nonpancreatic expression characteristic of each original founding mouse. The high pancreatic expression of the introduced rat genes is transcriptionally determined. In response to high rat elastase I mRNA content in the pancreas, rat elastase I protein is synthesized and secreted at high levels. The stable inheritance of expression of the foreign rat elastase I genes in transgenic mice allows the development of animal lines that synthesize and secrete high levels of foreign protein by the pancreas.

Animals↗
PubMed1985

Kallikrein-related mRNAs of the rat submaxillary gland: nucleotide sequences of four distinct types including tonin.

We 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 S1 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 S2 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.

Amino Acid Sequence↗
PubMed1985

Tissue-specific expression of kallikrein-related genes in the rat.

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.

Animals↗
PubMed1984

Structure of two related rat pancreatic trypsin genes.

A family of approximately 10 trypsin genes was detected in a rat genomic library by hybridization and in vivo recombination techniques using cloned rat pancreatic trypsin I and II cDNAs as probes. Two separate clones containing the entire trypsin I gene and most of the trypsin II gene were sequenced. Four introns split the trypsin I coding sequence. The positions of the first three introns of the trypsin II gene are identical with those in the trypsin I gene (the fourth intron was not present in the trypsin II clone). The coding regions of the two genes are 88% homologous; the 5'-noncoding regions are 92% homologous, whereas the 3'-noncoding regions share 66% identity. In contrast, the proximal 5'-flanking regions from -1 to -500 which may contain the elements controlling gene expression are less than 30% conserved overall, but segments of approximately 70% homology can be discerned in this region. Some of these sequences are homologous to sequences found in the chymotrypsin and elastase genes. More distal upstream sequences (-500 to -2500) and the intervening sequences show no evident sequence homology (less than 20%). Unique sequences containing homopolymeric purine/pyrimidine repeats are found 2.5 kilobases upstream from the start of transcription of the trypsin I gene and within the second and third introns of the trypsin II gene. The nucleotide homologies as well as the similarities of intron positions of the two trypsin genes to those of other serine protease genes clearly support an evolutionary relationship between members of this gene family.

Animals↗
PubMed1984

Structure of the two related elastase genes expressed in the rat pancreas.

We have isolated and characterized rat genomic DNA fragments bearing the two secretory elastase genes that are expressed in the exocrine pancreas. The complete exonic sequences for each of the genes as well as considerable intronic and flanking sequences are reported. Each elastase gene is interrupted by seven intervening sequences which are located at corresponding positions within the two genes, with one exception: the third intron of the elastase II gene has shifted one codon in the 5' direction. The placement of introns within the amino acid coding domains in part may reflect the formation of the progenitor serine protease gene by the duplication of an exon encoding a characteristic polypeptide structure comprising three beta sheets. The activation peptides of the zymogens and the signal peptides, which form discrete functional domains in the protein precursors, are encoded by separate exons. In addition to the TATAA box, the two genes share considerable sequence similarity in the 5'-proximal flanking regions (up to approximately 450 base pairs upstream); however, a number of gaps must be introduced to optimize the sequence alignment. The similarities are largely confined to six oligonucleotide regions with greater than 70% sequence conservation. The elastase I gene has a perfect repeating copolymer (GT)24 located 427-379 nucleotides upstream from the start of transcription. The elastase II gene has a similar GT-rich region (52/55 G or T) located 384-330 nucleotides upstream. Comparison of the 5'-flanking regions of the two elastase genes with those of pancreatic chymotrypsin and trypsin I and II reveals that one of the six conserved oligonucleotide regions is generally conserved for these genes as well. This conserved region contains putative enhancer core sequences.

Amino Acid Sequence↗
PubMed1984

Chromosomal assignments of human genes for serine proteases trypsin, chymotrypsin B, and elastase.

The genes for the serine proteases trypsin, chymotrypsin B, and elastase were chromosomally assigned in man using cDNA probes that have been isolated from a rat pancreatic cDNA library. DNA from human X rodent somatic cell hybrids was cleaved with BamHI or EcoRI and analyzed by Southern filter hybridization methods for the segregation of the genes for trypsin-1 (TRY1), chymotrypsin B (CTRB), and elastase-1 (ELA1). TRY1 was assigned to human chromosome 7q22----qter, CTRB to chromosome 16, and ELA1 to chromosome 12. Although the three genes are members of the same gene family, they are dispersed over different chromosomes.

Biological Evolution↗
PubMed1984

Chromosomal assignments of genes for trypsin, chymotrypsin B, and elastase in mouse.

The mouse genes for the serine proteases trypsin (Try-1), chymotrypsin B (Ctrb), and elastase (Ela-1) were chromosomally assigned using Southern blot hybridization of mouse X Chinese hamster cell hybrid DNA. cDNA probes for the three genes were hybridized to cell hybrid DNA cleaved with BamHI or HindIII and the segregation of Try-1, Ctrb, and Ela-1 was correlated with the segregation of mouse chromosomes. Try-1 is located on chromosome 6, Ctrb is on chromosome 8, and Ela-1 is on chromosome 15. The three genes fall into three syntenic groups that are conserved in the mouse and human genomes.

Animals↗
PubMed1984

Synthesis of single-stranded hybridization probes from reusable DNA templates bound to solid support.

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.

Bacteriophages↗
PubMed1984

Tissue-specific expression of the rat pancreatic elastase I gene in transgenic mice.

The gene for rat pancreatic elastase I is selectively expressed to high levels in the rat exocrine pancreas. When the cloned rat elastase I gene with 7 kb upstream and 5 kb downstream flanking sequences was introduced into mice by microinjection into fertilized eggs, the gene was expressed in a pancreas-specific manner. In four of five transgenic mice, the level of rat elastase I mRNA in the pancreas was equal to or greater than the normal rat level (10,000 mRNAs per cell) and correlated with the number of integrated gene copies. In nonpancreatic tissues the levels were at least 10(3)-fold lower, except for expression in the liver of one mouse. Thus transfer of a 23 kb genomic DNA segment containing the rat elastase I gene to a foreign chromosomal location in the mouse can give rise to qualitatively and quantitatively normal expression.

Animals↗
PubMed1984

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts approximately equal to 425 and approximately equal to 950 base pairs long, respectively, which are specific for bovine cholesterol side-chain 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 approximately equal to 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.

Adrenal Cortex↗
PubMed1982

Rat pancreatic ribonuclease messenger RNA. The nucleotide sequence of the entire mRNA and the derived amino acid sequence of the pre-enzyme.

We have cloned via recombinant DNA technology the mRNA sequence of rat pancreatic ribonuclease, and have determined the entire nucleotide sequence of the mature message. Clones bearing RNase sequences within a double-stranded complementary DNA library of rat pancreatic mRNA were initially detected by hybridization with size-fractionated rat pancreatic polyadenylated RNA that included mRNA 0.85 to 1.0 kilobase in length. Recombinant plasmids bearing RNase mRNA sequences were conclusively identified by comparison of the amino acid sequence of the encoded protein with the known amino acid sequence of rat RNase. RNase mRNA is 783 nucleotides in length, plus a poly(a) tail with an average length of 140 nucleotides, and contains long 5' and 3' noncoding regions relative to other pancreatic mRNAs. It encodes a secretory preRNase of 152 amino acid residues including a signal peptide of 25 amino acids.

Amino Acid Sequence↗
PubMed1982

Two similar but nonallelic rat pancreatic trypsinogens. Nucleotide sequences of the cloned cDNAs.

We have cloned and identified mRNA sequences for two rat pancreatic trypsinogens. Nucleotide sequence analysis of the cloned sequences revealed two mRNAs that encode similar, though noallelic, pretrypsinogens. Trypsinogen I mRNA is 804 nucleotides in length, plus an estimated poly(A) tract of 100 nucleotides, and contains a short (13 nucleotide) 5' noncoding region and a 3' noncoding region of 54 nucleotides. It encodes a preproenzyme of 246 amino acids comprising a hydrophobic prepeptide (signal peptide) of 15 amino acids, an activation peptide characteristic of trypsinogens, and an active form of trypsin, 223 amino acids in length, that has 78% amino acid sequence identity with porcine trypsin. Trypsinogen II mRNA has a nucleotide sequence 88% homologous with that of trypsinogen I mRNA and encodes a protein with 89% amino acid sequence identity with trypsinogen I. The enzymes encoded by trypsinogen I and II mRNAs retain the key amino acid residues that determine the characteristic substrate cleavage preference of trypsins and, therefore, represent the rat counterparts of this digestive enzyme. Trypsinogen I mRNA is a major pancreatic mRNA comprising an estimated 2-5% of the total, whereas trypsinogen II mRNA is present at much lower levels.

Alleles↗
PubMed1982

Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences.

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.

Amino Acid Sequence↗
PubMed1982

Identification of a cholesterol-regulated 53,000-dalton cytosolic protein in UT-1 cells and cloning of its cDNA.

UT-1 cells, a clone of Chinese hamster ovary (CHO) cells, have a 100- to 1,000-fold elevation in the amount of 3-hydroxy-3-methylglutaryl CoA reductase and therefore grow in the presence of compactin, an inhibitor of reductase. In this paper, we report that UT-1 cells also have a markedly increased amount of another protein with a Mr of 53,000 and an isoelectric point of approximately equal to 6. Whereas the reductase is an enzyme of the endoplasmic reticulum, the 53,000-dalton protein (termed the "53k" protein) is in the cytosol. It is not precipitated by an antireductase antibody. Synthesis of the 53k protein, like that of the reductase, is suppressed when UT-1 cells are incubated with plasma low density lipoprotein (LDL). We prepared a library of recombinant plasmids containing double-stranded cDNAs from UT-1 cells. Using differential colony hybridization, we identified recombinant plasmids containing double-stranded cDNA inserts encoding mRNAs expressed at high levels in UT-1 cells as compared with CHO cells. One of the plasmids, designated p53k-3, contained a 0.97-kilobase double-stranded cDNA that hybridized to a 3.8-kilobase mRNA. When translated in vitro, this 3.8-kilobase mRNA directed the synthesis of a protein identical to the cellular 53k protein as determined by two-dimensional gel electrophoresis. Hybridization studies showed that the mRNA for the 53k protein was present in much larger amounts in UT-1 cells than in parental CHO cells. In both cell types, the content of this mRNA decreased markedly when the cells were incubated with LDL. Although the function of the 53k protein is not known, circumstantial evidence suggests that it may represent cytosolic 3-hydroxy-3-methylglutaryl CoA synthase, the enzyme preceeding the reductase in the cholesterol biosynthetic pathway. The current data indicate that the synthesis of at least two proteins, the reductase and the 53k protein, are induced to high levels in compactin-resistant UT-1 cells and that the synthesis of both is suppressed coordinately by LDL.

Cells, Cultured↗
PubMed1982

Rat pancreatic kallikrein mRNA: nucleotide sequence and amino acid sequence of the encoded preproenzyme.

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'-noncoding 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 gamma 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.

Amino Acid Sequence↗
PubMed1982

Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme a reductase and evidence for regulation of its mRNA.

A recombinant plasmid containing a 1.2-kilobase cDNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated from a cDNA library prepared from UT-1 cells, a clone of Chinese hamster ovary cells that has markedly elevated reductase activity. This plasmid, designated pRed-10, was identified by differential colony hybridization and hybrid-selected mRNA translation. The mRNA that hybridized to pRed-10 directed the synthesis in vitro of a 90,000-dalton protein that was immunoprecipitated by an antireductase antibody. The same 90,000-dalton protein was immunoprecipitated when UT-1 cells were pulse labeled with [35S]methionine in vivo and rapidly solubilized with boiling NaDodSO4. By blot hybridization, pRed-10 hybridized to mRNAs of 4.2 and 4.7 kilobases in UT-1 cells. Both mRNAs were reduced to undetectable levels when low density lipoprotein, a suppressor of the reductase, was present in the culture medium. These data indicate that the primary translation product of reductase mRNA is a 90,000-dalton protein and that LDL suppresses the reductase in UT-1 cells by drastically reducing the level of its mRNA.

Animals↗