Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase isoforms in murine tissues determined by real-time PCR: a new view of a large family

William W. Young, Jr.1,2, Dana R. Holcomb2, Kelly G. Ten Hagen3 and Lawrence A. Tabak3

2 Department of Molecular, Cellular, and Craniofacial Biology, School of Dentistry and Departments of Biochemistry and Molecular Biology and Pharamacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40292
3 Biological Chemistry Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Received on January 19, 2003; revised on February 25, 2003; accepted on February 26, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family transfer GalNAc to serine and threonine sites and initiate mucin-type O-glycosylation. There are at least 13 functionally characterized family members in mammals. Explanations for the large size of this enzyme family have included functional redundancy, differences among isoforms in substrate specificity, and specific expression of individual isoforms in particular tissues or during certain developmental stages. To date no quantitative comparison of the levels of all ppGaNTase isoforms in any tissue of any species has been reported. We performed real-time polymerase chain reaction using the Taqman method to determine the expression of ppGaNTase isoforms in mouse tissues. Several tissues exhibited a common pattern in which isoforms T1 and T2 were the most strongly expressed, although the level of expression varied widely among tissues. In striking contrast to this general pattern, testis, sublingual gland, and colon exhibited distinctive profiles of isoform expression. Isoform T13 was expressed most strongly in brain, and one putative isoform was expressed only in testis. In mammary tissue the expression of several isoforms changed markedly during pregnancy and lactation. In summary these real-time PCR data indicate the contribution of each isoform to the overall ppGaNTase expression within each tissue and highlight the particular isoforms and tissues that will be the targets of future studies on the functions of the ppGaNTase family.

Key words: glycosyltransferases / mucins / O-linked glycosylation / real-time PCR


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family transfer GalNAc to serine and threonine sites to initiate mucin-type O-glycosylation. O-linked glycans are involved in numerous biological processes, such as leukocyte homing (Somers et al., 2000Go; Yeh et al., 2001)Go, function as ligands for receptors (Hooper and Gordon, 2001Go) and as signals for protein sorting (Alfalah et al., 1999Go), and can affect the coil-to-beta structural transition of the prion peptide (Chen et al., 2002Go). Despite the seeming simplicity of the reaction catalyzed by this family, at least 13 family members have been functionally characterized in mammals, and in silico analysis of public and private databases suggests there may be as many as 24 isoforms in humans (Ten Hagen et al., 2003Go). The need for such a large family has been explained partially by differences in substrate specificity, functional redundancy, and differences in expression patterns in terms of tissue specificity, cell-type specificity, or developmental stage specificity (reviewed in Ten Hagen et al., 2003Go). To date genetic ablation in mice of individual isoforms has failed to reveal unique functions for isoforms T1, T4, T5, and T13 (Ten Hagen et al., 2003Go). However, mutation of one family member imparted a lethal phenotype in Drosophila (Ten Hagen and Tran, 2002Go; Schwientek et al., 2003Go).

Despite the considerable investigative effort expended concerning the size and expression patterns of the ppGaNTase family, so far no quantitative comparison of the levels of all ppGaNTase isoforms in any tissue of any species has been made. Such a comparison cannot be made by enzyme activity assay because substrate specificities of the isoforms are largely overlapping. Within a cell the ppGaNTase activity for any given substrate will be the sum of the activities for all isoforms present, and the contribution from any one isoform cannot be determined. Therefore, in the present article we determined the expression of ppGaNTase isoforms at the level of mRNA by real-time polymerase chain reaction (PCR) using the Taqman method. The results demonstrate that many tissues share a common pattern of having isoforms T1 and T2 as the most highly expressed family members, whereas a few other tissues have expression patterns unique to those tissues. One putative isoform designated here as isoform Tc is expressed only in the testis. Finally, the expression of several isoforms varies during pregnancy and lactogenesis in mammary tissue.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Profiles within individual mouse tissues of ppGaNTase isoform transcripts
Whereas individual isoforms have been analyzed previously for expression in a variety of tissues by northern blotting, a quantitative comparison of expression of all isoforms within any tissue has not yet been reported. Therefore, we determined the expression levels of the mouse ppGaNTase isoforms available in the public database by real-time PCR (Figure 1). These data provide a profile of all isoform expression within each tissue so that the rank order of isoform expression can be visualized. There is a group of tissues (referred to as group A) in which isoforms T1 and T2 constituted a very high percentage of the total ppGaNTase transcript expressed. Group A includes brain, heart, liver, lung, muscle, ovary, prostate, spleen, thymus, and thyroid. For example isoforms T1 and T2 constituted 91% and 83% of total isoform expression in liver and heart, respectively, and in skeletal muscle T1 and T2 were the only isoforms with detectable expression. The ratio of expression of isoforms differed among group A tissues with T1 being greater than T2 in heart, liver, lung, muscle, spleen, and thymus; T2 being greater than T1 in brain, prostate, and thyroid; and both isoforms being similar in ovary. Within group A tissues the other isoforms exhibiting strong expression differed; that is, the next most strongly expressed isoform after T1 and T2 was putative isoform Ta in brain, isoform T8 (White et al., 2000Go) in lung, and isoform T10 (Ten Hagen et al., 2001Go) in ovary, prostate, and spleen.




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Fig. 1. Expression of ppGaNTase isoforms in mouse tissues. Taqman real-time PCR results are expressed as 1/(fold difference); fold difference is the difference between the experimental isoform value and GAPDH (see Materials and methods). Values are the mean of duplicate Taqman data points; in all cases the range between duplicate values was <3.8% of the mean. SLG, sublingual gland; muscle means skeletal muscle. Kidney data are from the kidney of a C3H strain female (see Table I). Group A tissues are identified by an asterisk (see text).

 

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Table I. ppGaNTase isoform expression in kidneys of males and females from various strains

 
In striking contrast to the pattern seen in group A tissues, the patterns of isoform expression in testis, sublingual gland, and colon were each distinctive. The isoform most strongly expressed in mouse testis was not isoform T1 or T2 but instead was isoform T3 (Figure 1). The expression of putative isoform Tb was remarkable because not only was it strongly expressed in testis, but also testis was the only tissue with detectable expression of that isoform. In addition isoform T10 was expressed strongly in the testis, making it the fourth most strongly expressed isoform, ahead of isoform T2 in the rank order. The expression pattern in the sublingual gland was also distinctive in that the rank order of expression (from stronger to weaker) was isoform T7 > T4 > T3 > T12 > T2 > T10 > T5 > T1. In comparison to the dominance of isoforms T1 and T2 in liver, heart, and skeletal muscle mentioned, in the sublingual gland isoforms T1 and T2 constituted only 13% of the total ppGaNTase transcript expressed. The colon as well had a distinctive pattern not dominated by isoforms T1 and T2 with the rank order being isoform T4 > T7 > T12 > T2 {approx} T10 {approx} T3 > T1 > T5.

Comparison among mouse tissues of ppGaNTase isoform expression
As described in the Discussion, we used glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as internal reference for all of the real-time PCR results reported here despite the fact that GAPDH expression varies under certain circumstances and may also vary between tissues. The following comparisons of expression levels among tissues must be viewed with that potential caveat in mind. Isoforms T1 and T2 were expressed in all tissues tested (Figure 1), confirming the ubiquitous expression found in earlier northern blotting studies and in recent real-time PCR analyses of isoform T1 in human tissues (Zhang et al., 2003Go). However, the expression of isoform T1 differed widely among tissues of group A with the expression in spleen being more than 10-fold greater than that in heart, muscle, or thyroid. Interestingly, isoforms T1 and T2 were the most strongly expressed isoforms only in group A tissues. The highest expression of any isoform in any tissue was that of isoform T3 in testis.

Recently isoform T13 was cloned, shown to be expressed most strongly in human nervous tissue, and found to synthesize Tn antigen (Zhang et al., 2003Go). Similarly, we detected isoform T13 expression most strongly in brain (1/fold expression=8.6x10-4; Figure 1), whereas the levels in lung and colon were 8-fold and 42-fold lower, respectively, than in brain, and the level in the other tissues was undetectable. Interestingly, the level of isoform T13 in brain was actually very low compared to the expression of the other isoforms; that is, isoform T13 ranked ninth when the isoforms were ranked from strongest to weakest, and the level of the isoform most strongly expressed in the brain, T2, was 22-fold greater than isoform T13. Nevertheless, this example illustrates the point that the expression of an individual isoform that bears a particular enzymatic specificity may be of functional importance (Zhang et al., 2003Go) even if its actual level of expression is quite low.

Adding the individual expression values for each isoform produces a value for the total expression of all ppGaNTase isoforms in each tissue. These total expression values differed greatly among tissues. The strongest total expression was found in testis (1/fold expression=0.47) followed by sublingual gland (0.31), lung (0.24), spleen (0.18), prostate (0.15), and colon (0.1). With the exception of the spleen, these tissues with strong total expression either had glandular components or contained actively secreting cells, such as the goblet cells of the lung and colon. Among the tissues with lower total expression, all were in group A, all had T1 and T2 as the most strongly expressed isoforms, and none were glandular except for the thyroid.

Expression patterns in mammary tissue
The preceding analysis of constitutive ppGaNTase isoform expression among mouse tissues is important for establishing baseline patterns and for identifying tissue-specific isoforms. However, additional information relevant to isoform function can be gained by comparing isoform expression under different physiological conditions. As an example of this approach, we analyzed mammary tissue because of its robust mucin production and because of the major changes that occur during pregnancy and lactation. The pattern in virgin mammary gland (Figure 2) was similar to that found in the group A tissues, with the two most active isoforms being isoforms T1 and T2 and with the next most prominent isoform being T8 as in the lung. During pregnancy, the expression of isoform T3 increased steadily, reached a final value at day 19 that was 6.9-fold greater than the virgin level and then decreased toward the virgin level during lactation and involution. Isoform T10 increased to a lesser extent during pregnancy and then continued to increase during lactation so that by day 10 of lactation the level was 7.7-fold greater than virgin level. During involution T10 expression also decreased toward the virgin level.



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Fig. 2. Expression of ppGaNTase isoforms in mammary tissues. Taqman real-time PCR results are expressed as 1/(fold difference) as in Figure 1. Key: tissues were harvested on days 5, 14, and 19 of pregnancy; days 1 and 10 of lactation; and days 1 and 4 of involution. There was no detectable expression of isoforms T6, T13, T14, and Tb in any mammary tissues. Values are the mean of duplicate or triplicate Taqman data points; the range between replicates was <2.5% of the mean.

 
Isoform T2 and putative isoform T8 showed a pattern opposite to that of T3 and T10. Isoform T2 decreased sixfold by the end of pregnancy, remained at that level during lactation, and then returned partway to the virgin level during involution. Isoform T8 decreased 2-fold by the end of pregnancy, remained at that level during the start of lactation, but by day 10 of lactation had decreased 19-fold below the virgin level. During involution T8 expression returned partway to the virgin level.

Comparison of ppGaNTase isoform expression among mouse strains
The preceding data were obtained with tissues from several strains of mice (see Materials and methods). Therefore, we sought to determine if there were differences in expression of ppGaNTase isoforms among mouse strains. For this purpose we analyzed the expression in kidneys from representative strains which differ at the major histocompatibility complex—namely, Balb/c (H2-d), C3H (H2-k), and C57/BL (H2-b). As with many other tissues described, isoform T1 was the most highly expressed isoform in the kidney (Figure 1). However, the most distinctive feature of ppGaNTase expression in the kidney was that isoform T11 was the second most highly expressed isoform. This finding is consistent with the recent report of Schwientek et al. (2002)Go, who found T11 to be most highly expressed in both human and mouse kidney among tissues screened by northern blotting. Isoform T11 was also expressed in lung and prostate about fivefold less strongly than in kidney (Figure 1).

Interestingly, the only statistically significant difference in isoform expression between strains was for isoform T11 between Balb/c and C57/BL mice (Table I). Expression of T11 was almost twice as great in the Balb/c kidneys as compared to C57/BL (Table I), whereas the level in C3H kidneys was intermediate between the other two strains. This difference between strains was apparent in kidneys from both males and females. The causes of these strain-specific differences in T11 expression remain to be determined. In summary we conclude that the expression of the ubiquitously expressed isoforms 1 and 2 (Figures 1 and 2) does not vary between strains nor does the expression of the strongly expressed isoforms T3, T8, and T14. However, the example of isoform T11 indicates the need for future studies of other tissues to test for strain differences in expression of the other isoforms that are not prominent in the kidney and to determine the causes for the strain-specific expression of isoform T11.

A striking finding of the analysis of isoform expression in the kidney was that the expression of isoform T1 differed depending on gender (Table I). In all three strains expression was stronger in males than in females although the difference was statistically significant only in the C3H strain and when the data from all three strains were combined (Table I). The basis for these gender-specific differences in expression will require additional analysis, but a precedent may be the androgen responsiveness of mouse kidney beta-glucuronidase (Thornton et al., 1998Go). However, because other tissues and RNAs used in the present study were pools from both males and females, except for the gender-specific tissues, this gender difference was not pursued in the present study.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this article we present the first comparison of the expression of all ppGaNTase isoforms in mouse tissues by real-time PCR. The results provide the opportunity to evaluate the contribution of each isoform to the total expression of ppGaNTases in each tissue. Previously it was only possible to compare northern blots of each isoform and to get at best a qualitative measure of the relative expression of isoforms within a tissue.

One striking finding is the contrast between the large group of tissues, known as group A here, that exhibit a relatively simple pattern of isoform expression and the few tissues, namely, colon, kidney, sublingual gland, and testis, with a much more complex pattern (Figure 1). The common pattern of group A tissues is that isoforms T1 and T2 are the predominant isoforms (Figure 1). These two isoforms have been proposed to serve "housekeeping" ppGaNTase functions partly because of their ubiquitous expression. However, immunohistochemistry with isoform-specific antibodies revealed that T1 and T2 were expressed in connective tissue cells of some tissues (Mandel et al., 1999Go), raising the possibility that ubiquitous expression by RNA analysis of whole organs may be due to cell-specific expression within cells found in all tissues. This point illustrates two limitations of real-time PCR analysis of isoform expression in tissues. First, assays of tissues do not reflect the relative expression profiles in specific cell types, thus necessitating the localization of expression within tissues by in situ hybridization. Second, demonstration of the actual production of the protein product must be revealed by immunohistochemistry. Both of these lines of study will be pursued in future investigations.

The complex patterns of isoform expression in colon, kidney, sublingual gland, and testis may reflect the complex architecture of those tissues with certain isoforms being expressed in different cell types. An additional or alternative explanation may be the need in those tissues for the synthesis of a variety of O-linked glycans. The fine structural specificity of the ppGaNTase isoforms is only partially understood (Hassan et al., 2000Go; Ten Hagen et al., 2003Go). In contrast to isoforms T1 and T2, which accept a large variety of substrates, isoforms T7 and T10 glycosylate glycopeptide substrates but not nonglycosylated substrates (Ten Hagen et al., 1999Go, 2001Go). Interestingly, both of these isoforms are elevated in colon and sublingual gland. Also, several isoforms prefer substrates of defined amino acid sequence (Hagen et al., 1997Go; Bennett et al., 1998Go, 1999Go; Ten Hagen et al., 1998Go), and certain substrates may require a particular isoform for their glycosylation (Iwasaki et al., 2003Go; Zhang et al., 2003Go). Thus, it is not surprising that tissues with ample mucin production, such as colon, sublingual gland, and testis, might require high-level expression of multiple ppGaNTase isoforms. However, additional data on the precise specificities of all isoforms must become available before we can fully interpret the results of isoform expression reported here.

Genetic ablation of individual ppGaNTase isoforms in mice so far has not produced recognizable phenotypes (reviewed in Ten Hagen et al., 2003Go). This failure may be due to functional redundancy among isoforms, suggesting that the knockout of multiple isoforms may be necessary. In this regard the real-time PCR results may be very useful because they provide a picture of relative expression levels of isoforms not previously available. For example, isoforms T1 and T2 constitute nearly all of the ppGaNTase expression in some group A tissues (Figure 1). Therefore, these findings lead to the prediction that a double knockout of T1 and T2 may answer the question as to whether ppGaNTase activity is essential in those tissues for viability and/or for proper development. The isoform expression results should also be useful during the analysis of knockout mice because the data focus attention onto those tissues showing strongest expression of each isoform.

Similarly, the results also identify the potential tissue distribution of several putative isoforms. The most obvious example is putative isoform Tb, which is expressed strongly and exclusively in the testis. Putative isoform Ta is expressed most strongly in brain, lung, and testis and putative isoform Tc in lung and ovary. Future efforts will be directed at determining which of these putative isoforms encode functional enzymes.

All expression data are based on the difference of each experimental value from the value for the internal reference, GAPDH, in that tissue. Although GAPDH is frequently used for this purpose, its expression is dependent on numerous factors (see reviews Suzuki et al., 2000Go; Bustin, 2001Go) and therefore may vary among tissues. As one means of assessing the present results independently of GAPDH, we compared our real-time PCR results with published northern blot results. There was generally a good correlation between the two data sets for isoforms 3 (Zara et al., 1996Go), 4 (Hagen et al., 1997Go), 5 (Ten Hagen et al., 1998Go), 7 (Ten Hagen et al., 1999Go), 10 (Ten Hagen et al., 2001Go), 11 (Schwientek et al., 2002Go), 12 (Guo et al., 2002Go), 13 (Zhang et al., 2003Go), and 14 (Wang et al., 2003Go). For isoforms T1 and T2 northern blot analysis showed ubiquitous expression in rat and mouse tissues (Hagen et al., 1997Go; Ten Hagen et al., 2001Go), thus making it difficult to compare those results quantitatively with the present real-time PCR data. Northern analysis data for human isoform T6 (Bennett et al., 1999Go) and putative human isoform T8 (White et al., 2000Go) did not correlate with the present real-time PCR data.

The results of isoform expression in mammary glands (Figure 2) provide an example of how physiological conditions may influence the expression profile of ppGaNTase isoforms. The virgin mammary gland consists of adipocytes with the ductal epithelium dispersed through the fat pad (reviewed in Hennighausen and Robinson, 2001Go; Shillingford and Hennighausen, 2001Go). During pregnancy the epithelium proliferates and differentiates under the influence of progesterone, placental lactogens, and prolactin. During lactation the ducts form side branches under the continued influence of prolactin, resulting in the secretory epithelium's completely filling the fat pad. Finally, when suckling ceases, the epithelium undergoes apoptosis and tissue remodeling, a process that is reversible at day 1 and irreversible by day 4 of involution.

The results indicate that the expression of ppGaNTase isoform T3 increased during pregnancy and isoform T10 increased during both pregnancy and lactation, whereas isoform T2 decreased during pregnancy and putative isoform T8 decreased during both pregnancy and lactation. At present it is not known whether the expression of any of these four isoforms is regulated by hormones present in mammary tissue. An alternative to hormonal regulation of isoform expression is that isoform T2 is expressed in the epithelial cells, whereas isoform T8 is expressed in the adipocytes, and, therefore, their changes in level of expression simply reflect the nature of the mammary tissue at each stage. Information about the regulation of ppGaNTase gene expression is available only for isoform T3. Nomoto et al. (1999)Go found that T3 expression is regulated by multiple systems, including transcription factor binding sites and a stem-and-loop structure in epithelial gland cells. Thus, future studies will be directed at assessing the factors responsible for these dramatic changes in ppGaNTase isoform expression during mammary gland development.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Tissues and RNA
Prostate, thymus, and thyroid tissues from Swiss Webster mice were purchased from Pel-Freez (Rogers, AR). Kidneys were harvested from 6–8-week-old Balb/c, C3H, and C57/BL mice. Brain, colon, heart, ovary, skeletal muscle, sublingual gland, and testis were harvested from male and female Balb/c mice. Total RNA was prepared from mouse organs using Trizol (Invitrogen, Carlsbad, CA) as described by the manufacturer. Total RNA from mammary tissue of C57/BL mice was a gift from Drs. F. Le Provost and L. Hennighausen. Total RNA from lung, liver, and spleen of Swiss Webster mice was purchased from Ambion (Austin, TX). The quality and quantity of total RNA and the size of amplicons produced by each Taqman reaction were analyzed using the Agilent 2100 Bioanalyzer (Wilmington, DE). All primer-probe sets were found to produce a single band of the predicted size.

Quantitative mRNA analysis by real-time PCR
mRNA gene expression levels were determined after reverse transcription by real-time PCR using an Applied Biosystems PRISM 7700 Sequence Detection System (Foster City, CA). Total RNA was treated with DNase, and then cDNA was synthesized using random hexamers and the SuperScript first-strand synthesis kit (Invitrogen). Primers and fluorogenic probes were designed using Primer Express (version 1.5, Applied Biosystems) according to the rules of Livak (1999)Go. Because of large regions of homology among the members of the ppGaNTase family (Hagen et al., 1999Go), primer-probe sets were chosen in regions of low homology to avoid cross-reactions with other isoforms. Regions of homology were identified by ClustalX. Primers were prepared by G. Poy, NIDDK, NIH, or were purchased from MWG Biotech (High Point, NC). Probes were purchased from Geneprobe Technologies (Gaithersburg, MD).

All assays were performed with the single thermocycling protocol of 50°C, 2 min; 95°C, 10 min; and 40 cycles of 95°C, 15 s, followed by 60°C, 1 min. The amplification efficiency was determined for each primer-probe set (Table II) as previously described (Bustin, 2001Go). Briefly, Ct values were determined for a series of 10-fold dilutions of template and the results plotted as the log of the initial target copy number versus Ct. All sets produced slopes close to the theoretical ideal of -3.3. The set of primers and probe for the internal reference, GAPDH, was purchased from Applied Biosystems.


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Table II. Primers and probes for mouse ppGaNTase isoforms

 
The 16 mouse ppGaNTase isoforms analyzed here are present in the public database (www.ensembl.org). Twelve of these isoforms have been cloned and shown to produce catalytically active enzyme: isoform T1 (U73820), T2 (AF348968), T3 (NM_015736), T4 (U73819), T5 (AF049344), T6 (AJ133523), T7 (AF349573), T10 (BC016585), T11 (BC011428), T12 (AB078146; Guo et al., 2002Go), T13 (AB078142; Zhang et al., 2003Go), and T14 (AB078144; Wang et al., 2003Go); note that isoform T10 was originally designated isoform T9 (Ten Hagen et al., 2001Go) and recently redesignated isoform T10 (Ten Hagen et al., 2003Go). No murine ortholog for human isoform T9 (Toba et al., 2000Go) was found in the database and thus was not studied in the current work. The other isoforms are derived from expressed sequence tags and appear in the Ensembl database as follows: isoform T8 (ENSMUSG00000038296), which remains a putative isoform pending verification of its enzymatic activity (Ten Hagen et al., 2003Go), and putative isoforms Ta (AB045325), Tb (AK005605), and Tc (AK019470).

Taqman results produce a Ct (threshold cycle) at which the fluorescence produced by cleavage of the fluorogenic probe rises above a background (or so-called threshold) level. Experimental values were compared to those obtained for a primer-probe set specific for an internal standard, GAPDH, and a delta Ct value was obtained that was the difference in the Ct values between experimental and GAPDH. In all cases expression of GAPDH was greater than that of any ppGaNTase isoforms. Because each cycle is actually a doubling, the delta Ct values were converted to a fold difference between experimental and GAPDH (2 cycles=4-fold, 3 cycles=8-fold, etc.). The data are presented as 1/(fold difference) so that stronger expression is represented by a higher value in Figures 1 and 2.


    Acknowledgements
 
We thank L. Hennighausen and F. Le Provost for RNA; D. Hein and M. Doll for use of the Taqman machine; R. Proia, M. Kono, and H. Mizukami for valuable discussions and instruction; and Y. Imbert for excellent technical assistance.

1 To whom correspondence should be addressed; e-mail: wwyoun01{at}gwise.louisville.edu Back


    Abbreviations
 
GAPDH, glyceraldehyde-3-phosphate-dehydrogenasePCR, polymerase chain reaction; ppGaNTase, UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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