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R J MacDonald

Publications and source records attributed to R J MacDonald.

At least 37 records · Page 2Linked to original sources
PubMed1992

Tissue-specific expression of kallikrein family transgenes in mice and rats.

To define the regulatory strategy for the transcriptional control of the kallikrein multigene family, we analyzed the expression of several kallikrein/SV40 T-antigen (TAg) fusion genes in transgenic mice and rats. Kallikrein family members are normally expressed at a high level in the submandibular gland and are expressed in a wide range of tissues that vary among individual family members. A total of 1.7 kb of proximal 5'-flanking DNA from the tissue kallikrein gene (rKlk1) was sufficient to confer much of the correct tissue-specific pattern on a TAg reporter gene. TAg mRNA was detectable in tissues that normally express rKlk1 and TAg-induced tumors arose in brain and pancreas. However, absolute levels of transgene mRNA were very low relative to the expression of the normal endogenous tissue kallikrein gene. In particular, expression in the salivary glands, normally very high for endogenous rKlk1, was either low or absent. An intact rKlk1 transgene with extensive flanking DNA (4.5 kb 5' and 4.7 kb 3') and complete intragenic (4 kb) sequences was expressed similarly to the fusion transgene, demonstrating that regulatory elements necessary for comprehensively correct expression are not contained within these additional gene regions. Two additional kallikrein/SV40 fusion transgenes were derived from other family members, one from the rKlk2 gene, which encodes tonin, and another from the rKlk8 gene, which encodes a prostate kallikrein. Whereas the endogenous rKlk2 and rKlk8 genes normally are expressed at high levels in rat salivary glands, they were not expressed in the salivary glands as transgenes. The results for these transgenes of three different family members indicate that control elements that direct the particular nonsalivary gland expression pattern characteristic of each family member may be present within the proximal 5'-flanking region of each gene, whereas regulatory sequences necessary for normal levels of expression in these tissues and for maximal salivary gland expression are not. We propose that the gene-associated regulatory sequences are complemented by a dominant control region that imposes salivary gland expression on the extended kallikrein family locus.

Animals↗
PubMed1991

Evolution of the rat kallikrein gene family: gene conversion leads to functional diversity.

Kallikrein-like simple serine proteases are encoded by closely related members of a gene family in several mammalian species. Molecular cloning and genomic Southern blot analysis after conventional and pulsed-field gel electrophoresis indicate that the rat kallikrein gene family comprises 15-20 members, probably closely linked at a single locus. Determination of the nucleotide sequences of the rGK-3, -4, and -6 genes here completes sequence data for a total of nine rat kallikrein family members. Comparison of the rat gene sequences to each other and to those of human and mouse kallikrein family genes reveals patterns of relatedness indicative of concerted evolution. Analysis of nucleotide sequence variants in kallikrein family members shows that most sequence variants are shared by multiple family members; the patterns of shared variants are complex and indicate multiple short gene conversions between family members. Sequence exchanges between family members generate novel assortments of variants in amino acid coding regions that may affect substrate specificity and thereby contribute to the diversity of enzyme activity. Furthermore, small sequence exchanges also may play a role in generating the diverse patterns of tissue-specific expression of rat family members. These analyses indicate an important role for gene conversion in the evolution of the functional diversity of these duplicated genes.

Amino Acid Sequence↗
PubMed1991

The Patient-Focused Hospital: a patient care concept.

Steadily rising costs, increased competition, and employee and customer dissatisfaction have prompted hospitals to turn to a variety of traditional approaches to improving operations and performance. Extensive diagnostic analyses conducted in several hospitals have led Booz, Allen to conclude that these traditional approaches fall significantly short of providing lasting, substantial operations and performance improvement. As a result of these analyses, Booz, Allen has developed a new operational strategy known as the Patient-Focused Hospital. Implementation of this strategy at pilot sites has proven that it can improve significantly service performance as well as customer and employee satisfaction and reduce hospital operating costs. This article identifies the circumstance that gave birth to the Patient-Focused Hospital concept and describes how it works. The article also discusses the implications of patient-focused operations within the hospital industry and predicts that hospitals that adopt this strategy now will be the leaders of the future.

Health Facility Environment↗
PubMed1990

The expression of two kallikrein gene family members in the rat kidney.

The mRNAs for two kallikrein gene family members expressed in the rat kidney have been characterized. One mRNA (PS) has previously been found in the pancreas and submaxillary gland and encodes true kallikrein. The second mRNA (K1) encodes a novel kallikrein-like enzyme expressed in the kidney and submaxillary gland that retains many of the key amino acid residues for the characteristic enzymatic cleavage specificity of kallikrein. Two oligonucleotide hybridization probes specific for the K1 mRNA demonstrate that the K1 mRNA is expressed in the kidney and submaxillary gland, but in none of the other eight tissues known to express one or more members of the rat kallikrein gene family. The K1 mRNA is the dominant kallikrein-related mRNA of the kidney, expressed at roughly 10 times the level of the true kallikrein (PS) mRNA. In the submaxillary gland the K1 mRNA is expressed at roughly one-fourth the level of true kallikrein mRNA.

Amino Acid Sequence↗
PubMed1990

Oestrogen administration and the expression of the kallikrein gene family in the rat submandibular gland.

Using a series of oligonucleotide probes (18-21 mers) specific for members of the rat kallikrein/tonin (arginyl-esteropeptidase) gene family (PS, S1, S2, S3, K1, P1), we have shown by Northern blot analysis that all six genes are expressed in the submandibular gland (SMG), with PS (true kallikrein) the most abundant in both male and female rats. Though female levels of PS mRNA are similar to that in the male, levels of mRNA from both the kallikrein-like (S1, K1, P1) and tonin (S2)/tonin-like (S3) genes are all substantially lower in the female than in the male rat. In contrast with the oestrogen dependence of anterior pituitary kallikrein (PS) gene expression, oestrogen administration (6 micrograms/day for 8 days) to castrate male or female rats is without effect on PS or S1, S2, S3, K1, P1 mRNA levels in the SMG. These findings suggest a tissue-specificity in the oestrogen regulation of true kallikrein gene expression in the two tissues. In intact male rats, oestrogen administration lowers SMG levels of S1, S2, S3, K1, and P1 but not PS mRNA to castrate levels, presumably by suppression of the pituitary/gonadal axis, consistent with the previously reported androgen dependence of SMG expression of these genes with the exception of PS.

Animals↗
PubMed1990

Kallikrein messenger RNA in rat arteries and veins.

Glandular kallikrein (EC 3.4.21.8) belongs to a subgroup of serine proteases coded by a multigene family. A kininogenase resembling glandular kallikrein has been identified in vascular tissue; however, it is not clear whether it is synthesized by vascular tissue or taken up from plasma. To determine the potential for kallikrein synthesis in vascular tissues, we tested whether messenger RNA (mRNA) for glandular kallikrein is present in rat arteries and veins. Poly(A+) RNA was isolated from pools of arteries or veins (n = 3, 30 rats each). Poly(A+) RNA from the kidney and liver was used as a positive and negative control, respectively. As a probe, we used rat pancreatic kallikrein 32P-labeled complementary DNA, which recognizes mRNA of the entire rat kallikrein family. Slot-blot analysis indicated that kallikrein mRNA was present in mRNA from the arteries, veins, and kidney but not from the liver. Poly(A+) RNA from arteries and veins contained approximately 1% as much kallikrein mRNA as that from the kidney. To confirm the slot-blot results and determine whether the mRNA for true glandular kallikrein was present in vascular tissue, we employed a polymerase chain reaction assay, first using primers specific for the entire kallikrein family (which amplify a 430-bp fragment) and then using primers specific for true glandular kallikrein mRNA (which amplify a 370-bp fragment). After the polymerase chain reaction assay, both arteries and veins showed fragments of these sizes when tested with rat kallikrein complementary DNA probe, thus confirming the presence of glandular kallikrein mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Expression of two kallikrein gene family members in the rat prostate.

We have characterized two kallikrein gene family members expressed in the prostate and submaxillary glands of rats. One mRNA (S3) is identical with the previously characterized submaxillary gland S3 mRNA that encodes an enzyme closely related to tonin. The second mRNA (P1) encodes a novel kallikrein-like enzyme that retains key amino acid residues responsible for the characteristic enzymatic cleavage specificity of kallikrein. Two P1-specific oligonucleotide probes derived from the P1 mRNA sequence were used to demonstrate the presence of P1 mRNA in the prostate and submaxillary glands and its absence in eight other rat tissues known to express one or more members of the kallikrein family. The P1-coding gene (rGK-8) was identified among genomic clones containing kallikrein family members by hybridization with a P1-specific oligonucleotide probe. The identification of the P1 gene was verified by nucleotide sequencing; the exon sequences of rGK-8 match the P1 mRNA sequence. The upstream region of rGK-8, where transcriptional regulatory elements likely reside, is very similar to that of other rat kallikrein family genes which are expressed in distinct tissue-specific patterns.

Amino Acid Sequence↗
PubMed1989

Organization and expression of the rat kallikrein gene family.

We have cloned and characterized eight members of the kallikrein gene family from rat genomic DNA. Three of the cloned genes correspond to the previously characterized kallikrein family mRNAs PS, S2, and P1, which encode true kallikrein, tonin, and a novel kallikrein-like enzyme, respectively. In two cases, two kallikrein family genes are located on single genomic clones, suggesting close linkage of this family in the rat genome. Based on the total number of cloned genes and mRNAs, the minimum size of the rat family is 11 genes. Comparisons between the rat genes demonstrate a high degree of nucleotide sequence identity (greater than 80%) in exonic, intronic, and nearby flanking regions. This extensive sequence conservation not limited to clearly functional domains suggests that concerted evolution of this gene family has occurred. Despite the high overall sequence conservation among the rat family members, several discrete regions of high variability exist in the coding regions. We have defined the tissue-specific expression of the PS (true kallikrein), S2 (tonin), and S3 mRNAs with gene-specific oligonucleotide probes derived from these variant regions. PS is expressed in a wide range of tissues, while S2 mRNA is restricted to the submaxillary gland and S3 mRNA to the submaxillary and prostate glands. The high sequence conservation within the upstream flanking regions of these genes suggests that a small number of nucleotide differences determines the disparate transcriptional specificity of individual family members.

Amino Acid Sequence↗
PubMed1989

Differential requirements for cell-specific elastase I enhancer domains in transfected cells and transgenic mice.

The 134-bp enhancer region of the pancreatic elastase I gene is sufficient to direct pancreatic acinar cell-specific transcription in transgenic mice and in transfected cells in culture. Ten-base-pair scanner mutations in three separate enhancer domains that inactivate enhancer function in transfected pancreatic cells in culture have no significant effect in transgenic mice. Because any pair of the three domains is sufficient to direct pancreas-specific expression in mice, no one domain is required for pancreas-specific transcription. Disruption of any two domains does inactivate the enhancer function in transgenic mice. Therefore, the elastase I enhancer domains essential for function in transfected cells in culture are not essential in animals but have a redundant function not apparent in transfected cells. This redundant function is not because of the particular acinar cell line used for transfections, the nature of the reporter gene, or the state of integration of the foreign test gene. We conclude that a trans-acting transcription factor or a modification of a factor(s) present in pancreatic cells of an animal is absent in pancreatic acinar cell lines.

Animals↗
PubMed1988

Structural characterization of the rat carboxypeptidase A1 and B genes. Comparative analysis of the rat carboxypeptidase gene family.

Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified.

Amino Acid Sequence↗
PubMed1988

Androgen dependence of specific kallikrein gene family members expressed in rat prostate.

We have used oligonucleotide probes specific for members of the rat kallikrein/tonin gene family (PS, S1, S2, S3, K1, and P1) to establish which arginyl esteropeptidase (kallikrein-like) genes are expressed in the prostate. We have also compared the expression and androgen dependence of these genes in prostate, submaxillary gland (SMG) and kidney. Only S3 (tonin-like) and P1 (kallikrein-like) are expressed in the prostate, with S3 very much more abundant. Prostatic S3 mRNA disappears after 8 days castration and is restored to intact levels by dihydrotestosterone (DHT) but not estradiol benzoate (EB) for 8 days. Prostate P1 mRNA levels were similarly but not identically affected. All six genes are expressed in the SMG, with PS (true kallikrein) the most abundant. Levels of PS mRNA in SMG are unaffected by castration, DHT, or EB treatment, although mRNA levels of other kallikrein-like (S1, K1, and P1), tonin (S2), and tonin-like (S3) genes fall 40-60% after castration, and are unaffected or partially restored by DHT and/or EB administration. Only PS and K1 are expressed in the kidney, at much lower levels than in the SMG and unaffected by castration or steroids. These studies thus confirm and extend the concept of tissue specificity of arginyl esteropeptidase gene expression, and further demonstrate that the same gene(s) is differentially regulated by androgens in the rat prostate, SMG, and kidney.

Animals↗
PubMed1988

Kallikrein gene expression in estrogen-induced pituitary tumors.

Anterior pituitary kallikrein-like enzyme activity, immunoreactivity and mRNA levels have previously been shown to be regulated by estrogen, in parallel with prolactin. In this study, we have examined the relationship between kallikrein and prolactin mRNA levels in estrogen-induced pituitary tumors. Treatment of Fischer 344 rats with diethylstilbestrol implants for 3, 5 and 7 weeks produced a dramatic increase in kallikrein mRNA levels and a modest increase in prolactin mRNA levels. These changes were partially reversed by bromocriptine treatment, and completely reversed by bromocriptine plus estrogen withdrawal. Using a panel of oligonucleotide probes specific for various members of the rat kallikrein gene family, we have shown that the kallikrein-like gene expressed appears to be true kallikrein.

Animals↗
PubMed1988

Limited transcription of rat elastase I transgene repeats in transgenic mice.

The rat elastase I (EI) regulatory region is an enhancer that directs efficient pancreas-specific transcription of linked genes integrated in the chromosomes of transgenic mice. However, with increasing numbers of tandemly repeated EI transgenes, the transcription rate per gene decreased. This decrease was not due to a titration of essential transcription factors, because transgenic mice with as many as 250 copies of the rat EI enhancer continued transcription of the endogenous mouse EI gene at its normal rate. Furthermore, when transgenic mice bearing 250 EI genes and mice bearing 7 copies of an EI enhancer-directed human growth hormone (hGh) gene were mated to produce mice with two unlinked arrays of EI enhancer-driven transgenes, each array was transcribed at the same rate as in mice bearing each array separately. Long tandem arrays of transgenes may inhibit efficient transcription despite the presence of ample amounts of essential transcription factors. Although the transcription rate of the mouse EI gene was not affected by the presence of large numbers of transgenes, the amounts of mouse elastase I and elastase II mRNAs were decreased in the presence of high levels of transgene mRNA, indicating an adjustment of the cell mRNA population.

Animals↗
PubMed1988

The cell-specific elastase I enhancer comprises two domains.

Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.

Animals↗
PubMed1987

The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice.

A total of 134 base pairs of the 5' flanking sequence of the elastase I gene is sufficient and necessary to direct expression of the passive human growth hormone gene (hGH) to the exocrine pancreas. We demonstrate that this elastase I regulatory region contains a transcriptional enhancer which directs acinar cell-specific expression in transgenic animals. The elastase I enhancer specifies correct expression of the linked hGH gene in an orientation- and position-independent manner and can activate a heterologous promoter. The enhancer also directs the appropriate temporal activation of the hGH gene in the developing pancreas. Transcription is initiated correctly for the elastase I or hGH promoter, and the transcripts are correctly processed regardless of the enhancer position within or outside the fusion gene. The elastase I enhancer generates coincident DNase I-hypersensitive sites in pancreatic chromatin when moved 3 kilobases upstream or within the first intron of the hGH gene and when associated with the hGH promoter.

Animals↗