Biomedical subjects

R W McKenzie

Publications and source records attributed to R W McKenzie.

6 recordsLinked to original sources

PubMed2000

Quantification of MUC2 and MUC5AC transcripts in human conjunctiva.

PURPOSE: Transcripts of mucins 1, 4, and 5AC have been identified in human conjunctival tissue. Of these, only MUC5AC has been localized to goblet cells. MUC2 is a goblet cell mucin originally identified in the intestinal mucosa. The presence of MUC2 transcripts and levels of MUC2 and MUC5AC transcripts in normal human conjunctiva, as determined by quantitative polymerase chain reaction (PCR), is reported. METHODS: RNA from conjunctivae of six donors (3 men, 3 women, 44 to 69 years, all white) was isolated and subjected to competitive reverse transcription-PCR. Internal standards, which are dsDNA molecules with ends complementary to a given primer pair but containing nonhomologous central sequences, were prepared for each gene assayed. Titration of a constant amount of cDNA against serial dilutions of the internal standard allowed quantification of the template cDNA. MUC2 and MUC5AC levels were compared to levels of the "housekeeping" gene, beta2-microglobulin (beta2M). The identity of PCR products was confirmed by sequencing. RESULTS: In the six individual samples tested, beta2M mRNA is expressed, on average, at approximately 10(-20) moles per sample (1 microg RNA) or approximately 63.5x10(4) molecules. The mRNA encoding MUC5AC, a relatively abundant ocular mucin, exists at levels 10-fold lower than beta2M. In contrast to previous reports of MUC2 mRNA being absent at the ocular surface, these results show that MUC2 transcripts are present and expressed at levels 5900-fold lower than for MUC5AC. Apparently, MUC2 transcripts exist on the order of only approximately 100 to 1000 molecules/microg of RNA in the analyzed samples. CONCLUSIONS: MUC2 transcripts are present in human conjunctival tissue and their abundance is much lower than that of MUC5AC. This is the first application of competitive PCR to the quantitative analysis of mucin expression in human ocular tissue. The sensitivity of this method allows the detection of MUC2 transcripts that were not detected by Northern blot analysis or in situ hybridization in previous studies. It also makes possible the comparison of relative levels of expression for ocular mucins.

Adult↗

PubMed1999

MUC5AC mucin is a component of the human precorneal tear film.

PURPOSE: Mucins are important structural and functional components of the precorneal tear film, yet little is known of their composition and synthesis. The mRNAs of MUC1, MUC4, and MUC5AC have previously been identified in human conjunctiva. Of these, only MUC5AC mRNA appears to be associated with goblet cells. The purpose of the this study was to quantify MUC5AC transcript levels, to identify MUC5AC protein in conjunctiva, tears, and goblet cells and to determine whether this mucin is secreted in response to the calcium ionophore ionomycin. METHODS: MUC5AC mRNA from normal human conjunctiva was identified, quantified, and compared with beta2-microglobulin levels using a competitive reverse transcription-polymerase chain reaction (RT-PCR) method. An antibody to a MUC5AC peptide was used to localize this mucin in conjunctival sections by immunohistochemistry. Anti-MUC5AC antiserum was used to label western blot analysis of conjunctiva and tears. Conjunctival tissues were incubated with ionomycin, and secreted mucins were detected with Helix pomatia agglutinin conjugated to horseradish peroxidase and with anti-MUC5AC antiserum. RESULTS: MUC5AC and beta2-microglobulin transcripts were expressed at a ratio of approximately 1:500. Immunochemical labeling showed that MUC5AC was localized in conjunctival goblet cells and at the apical surface of the conjunctival epithelium. MUC5AC protein was present in conjunctiva and in the tear film. Ionomycin stimulation of conjunctival secretion resulted in a fourfold increase in total mucin secretion and in a corresponding increase in secreted MUC5AC. CONCLUSIONS: MUC5AC is synthesized by goblet cells of the normal human conjunctiva, and this mucin is a component of conjunctival secretions and of normal human tears.

Adult↗

PubMed1998

cis-Acting sequences controlling the adult-specific transcription pattern of the Drosophila affinidisjuncta Adh gene.

The cis-acting sequences required for the adult-specific expression pattern of the alcohol dehydrogenase (Adh) gene of the Hawaiian picture-winged fruit fly, Drosophila affinidisjuncta were analyzed by germline transformation. Normally this gene produces two developmentally regulated transcripts. The upstream (distal) promoter produces a distal transcript, which makes up about 80% of the total in adults, while the downstream (proximal) promoter produces a corresponding proximal transcript, which accounts for the remainder. Previously constructed genes lacking regions corresponding to regulatory elements within the Drosophila melanogaster Adh gene or regions known to be required for full expression of the D. affinidisjuncta Adh gene in larvae were analyzed by introduction into the germline of D. melanogaster followed by RNase-protection analysis of RNA levels. In addition, to test a model of preferential promoter utilization by which transcription at the proximal promoter is inhibited by transcription initiated at the upstream distal promoter, a construction lacking the distal promoter was analyzed. Sequences homologous to the adult enhancer of the Adh gene of D. melanogaster appear to play a similar role in the D. affinidisjuncta gene. In contrast to what has been reported for other Drosophila Adh genes, this and some other regulatory elements are shared by the two promoters of the D. affinidisjuncta gene. Taken together, the results favor a model of stage-specific switching between the two promoters of the D. affinidisjuncta gene that involves competition for limiting components stimulating transcription, rather than interference by read-through from the upstream promoter.

Alcohol Dehydrogenase↗

PubMed1998

Cis-acting sequences contributing to expression of the Drosophila affinidisjuncta Adh gene in both larvae and adults.

To characterize the cis-acting sequences required for expression of the alcohol dehydrogenase (Adh) gene of the Hawaiian picture-winged fruit fly Drosophila affinidisjuncta, germ-line transformation was used to introduce altered forms of this gene into Drosophila melanogaster. Genes were constructed lacking regions corresponding to known or putative regulatory elements within the D. melanogaster gene as well as to sequences previously shown to be required for expression of the D. affinidisjuncta gene in the larval fat body as assayed by transient transformation. Measurement of alcohol dehydrogenase (EC 1.1.1.1) activity levels produced by the transfected genes indicates that multiple elements in both the 5' and 3' regions of the gene contribute to expression. The dispersed elements appear to function redundantly to ensure high levels of expression. Moreover, in contrast to what has been reported for other Drosophila Adh genes, some of the regulatory elements influence expression in both larvae and adults. In total, these results reveal a regulatory system in which the transcribed region in imbedded in an extended genomic segment rich in cisacting regulatory information.

Alcohol Dehydrogenase↗

PubMed1996

The two small introns of the Drosophila affinidisjuncta Adh gene are required for normal transcription.

All Drosophila alcohol dehydrogenase (Adh) genes sequenced to date contain two small introns within the coding region. These are conserved in location and, to some extent, in sequence between the various species analyzed. To determine if these introns play a role in Adh gene expression, derivatives of the Drosophila affinidisjuncta Adh gene lacking one or both introns were constructed and analyzed by germline and transient transformation of Drosophila melanogaster. Removal of both introns lowered expression, whether measured by enzyme activity or by RNA levels. The decrease was seen in both germline transformed and transiently transformed larvae, with the effect being larger for germline transformants. Similar decreases (averaging 5-fold) were also seen at the embryonic and adult stages for germline transformants. Nuclear run-off transcription with nuclei from germline transformed embryos indicated that the reduction in RNA levels is due to decreased transcription. However, LacZ fusion constructs designed to test for the presence of a classical enhancer in the introns provided no evidence for such a mechanism. Removal of each intron individually resulted in more complex phenotypes. The introns have smaller, additive effects on expression in adults. In larvae, removal of the upstream intron significantly increases RNA levels but modestly decreases enzyme activity. Removal of the downstream intron lowers expression in both germline and transiently transformed larvae, but also increases position effects in germline transformants. Therefore, the small introns are clearly needed for optimal transcription of this Adh gene, but multiple mechanisms are involved.

Alcohol Dehydrogenase↗

PubMed1994

Redundant cis-acting elements control expression of the Drosophila affinidisjuncta Adh gene in the larval fat body.

The alcohol dehydrogenase (Adh) gene in the Hawaiian species of fruit fly, Drosophila affinidisjuncta, like the Adh genes from all Drosophila species analyzed, is expressed at high levels in the larval fat body via a larval-specific promoter. To identify the cis-acting elements involved in this highly conserved aspect of Adh gene expression, deleted D. affinidisjuncta genes were introduced into D. melanogaster by somatic transformation. Unlike previously described methods, this transformation system allows analysis of Adh gene expression specifically in the larval fat body. The arrangement of sequences influencing expression of the proximal promoter of this gene in the larval fat body differs markedly from that described for the Adh gene from the distant relative, D. melanogaster. Multiple redundant elements dispersed 5' and 3' to the gene, only some of which map to regions carrying evolutionarily conserved sequences, affect expression in the fat body. D. affinidisjuncta employs a novel mode of Adh gene regulation in which the proximal promoter is influenced by sequences having roles in expression of the distal promoter. This gene is also unique in that far upstream sequences can compensate for loss of sequences within 200 bp of the proximal RNA start site. Furthermore, expression is influenced in an unusual, context-dependent manner by a naturally-occurring 3' duplication of the proximal promoter--a feature found only in Hawaiian species.

Alcohol Dehydrogenase↗