Differential expression of NAT1 translational repressor during development of bovine intramuscular adipocytes

Kirby D. Childs1, David W. Goad2, Mark F. Allan3, Daniel Pomp3, Clinton Krehbiel1, Rodney D. Geisert1, J. Brad Morgan1 and Jerry R. Malayer2

1 Department of Animal Science, Oklahoma Agriculture Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078
2 Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078
3 Department of Animal Science, University of Nebraska, Lincoln, Nebraska 68683


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This study was undertaken to test for differential gene expression in intramuscular adipocytes during fat deposition of feedlot steers. Angus x Hereford steers (n = 50) were fed a high-energy concentrate ration ad libitum for 20 (n = 5), 86 (n = 15), 121 (n = 15), and 146 days (n = 15) to obtain various degrees of intramuscular adipocyte development. Carcass traits were significantly different (P < 0.05) between the groups. Intramuscular adipose tissue was excised from the longissimus dorsi and snap frozen in liquid nitrogen. Pooled samples of total RNA representing each group were analyzed by differential-display polymerase chain reaction using 200 primer combinations comprising 20 arbitrary (5') and 10 anchor (3') oligonucleotides. Bands (n = 70) representing putative differences among treatment groups were excised, sequenced, and subjected to BLAST homology search. From these, 40 contained significant homology to known genes. One was of particular interest, the translational repressor NAT1 (novel APOBEC-1 target-1). NAT1 mRNA was quantified in individual animals to confirm differential expression among treatment groups. Results indicate that NAT1 message is more abundant (P < 0.05) in intramuscular adipocytes of younger/leaner animals.

adipocyte; differential display; marbling


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
PRODUCTION INEFFICIENCIES in the beef industry account for a loss of approximately 7.4 billion dollars annually. Most of the inefficiencies are due to excessive external lipid deposition and inferior muscling; the United States produces over 2 billion kilograms of excess lipid each year (19). One reason for the production of excess lipid in beef cattle is that feeding diets high in starch (i.e., cereal grains) for extended periods of time improves the palatability and acceptability of meat for the US consumer (7). In fact, the current USDA (23) grading system, which is used to determine the quality and value of the product, revolves around the amount of intramuscular adipose tissue (marbling) found in a cross section of the longissimus dorsi muscle. Intramuscular adipose tissue not only plays a key physiological role in beef cattle but also affects the taste and tenderness of the final product.

Adipogenesis involves a specific series of events to initiate and maintain the differentiation pathway. Studies utilizing 3T3 cells have characterized early events in the differentiation of preadipocytes into adipocytes, including the expression profiles of two families of transcription factors induced in this early phase, the CCAAT/enhancer binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs). These transcription factors are responsive to various adipogenic inducers including insulin, dexamethasone, long-chain fatty acids, and retinoids (1, 3, 6, 11, 22, 24). A specific transcription profile ensues through preadipocyte to adipocyte differentiation, leading eventually to expression of genes associated with terminal differentiation of the adipocyte.

Because of the importance of intramuscular adipose development to the economics of beef production, we undertook this study to test the hypothesis that gene expression profiles in intramuscular adipose tissue change during the period of high grain feeding. The objective of this experiment was to use differential-display PCR (ddPCR) to examine changes in mRNA profiles in intramuscular adipose during stages of high grain feeding in beef steers to identify potential regulators and/or mediators of the development process.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Animals
Angus x Hereford steers (n = 50) were sampled from a ranch in western Oklahoma, where the herd has been selected for the propensity to deposit high amounts of intramuscular fat (i.e., marbling) and produce carcasses which grade "US Choice" or higher. Animals were raised, weaned, and preconditioned using standard commercial practices. At 12 mo of age, steers were blocked by weight, assigned to 10 pens of 5 steers each, and provided a standard high concentrate grain diet ad libitum at the Willard Sparks Beef Research Facility in Stillwater, OK. Animals were fed this diet for either 20, 86, 121, or 146 days to yield carcasses representing different levels of adipocyte deposition. Groups (days on the concentrated diet) were targeted to have 0, 0.7, 1.3, or 1.8 cm of subcutaneous fat at the 12th/13th rib interface at the time of harvest. Steers were routinely weighed, and ultrasound was used to monitor subcutaneous fat deposition during the feeding period. After the group average of steers (n = 15; 3 groups of 5) reached target subcutaneous fat levels, the animals were humanely killed at the Food and Agricultural Products Center in Stillwater, OK, and tissues were collected within 10 min postmortem. Following a 24- or 36-h chilling period (0–1°C), trained Oklahoma State University personnel collected carcass yield and quality grade factors consisting of hot carcass weight, longissimus muscle area, subcutaneous fat thickness, kidney, pelvic and heart fat percentage, skeletal and lean maturity, and marbling score.

Tissue Collection
Prior to dissection, work surfaces and tools were sterilized with a 10% Clorox solution. Longissimus dorsi sections (~250 g) were collected from the left side of each carcass. Each longissimus section was removed at the 13th rib using a sterile scalpel and immediately transferred to a 4°C processing room. Muscle cores were weighed and rinsed in sterile 0.9% saline and kept on ice for the duration of the dissection. Intramuscular adipose tissue (marbling) was dissected from the muscle core, and the surrounding muscle fibers were separated from the intramuscular fat. The adipose and muscle fiber samples were properly labeled and snap frozen in liquid nitrogen. Samples were stored at -80°C until extraction of RNA.

RNA Extraction and Preparation
Total RNA was extracted from 0.3–1 g of adipose tissue with 4 ml of TRIzol reagent (GIBCO; Life Technologies, Grand Island, NY) according to manufacturer’s instructions. Tissue samples were homogenized with a VirTishear homogenizer (Virtus, Gardiner, NY) for 45 s and allowed to incubate at room temperature for 5 min. RNA pellets were air dried, resuspended in 200 µl of Tris-chloride EDTA (TE) buffer, and stored at -80°C until needed. Following pooling (see below), RNA samples were treated with DNase I for removal of DNA contamination. Removal of DNase I was completed by using standard phenol/chloroform extraction procedures as described by Allan et al. (2). Concentrations of total RNA were quantified by in a fluorometer (model TD-700; Turner Designs, Sunnyvale, CA).

RNA pools for differential display.
To reduce the potential for false positives in ddPCR products, two replicates of each group were used for the ddPCR analysis to enable selection of bands demonstrating consistent expression profiles within treatment as described by Allan et al. (2). A replicate consisted of selecting RNA from five animals closest to the average mean marbling score (Table 2) in each harvest group. A total of 5 µg of RNA from each of the five animals in a replicate group was mixed together.


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Table 2. Performance and carcass traits of feedlot steers stratified by days on the concentrated diet

 
cDNA preparation.
Pooled samples of total RNA were reverse-transcribed to generate cDNA. One microgram of pooled total RNA was combined with Moloney murine leukemia virus reverse transcriptase (MMLV-RT, 200 U; Promega), oligo(dT) primer (1.0 µg; Promega), dNTPs (0.5 mM each; Promega), Tris·HCl (50 mM; pH 8.3), KCl (75 mM), MgCl2 (3 mM), dithiothreitol (10 mM), and RNasin (20 U; Promega). The reaction mixture was incubated at 22°C for 15 min followed by incubation at 42°C for 30 min, and the reaction was terminated by heating at 95°C for 5 min and quickly cooling to 4°C

cDNA quality control.
To establish that the cDNA made from RNA pools was of high quality and consistency, it was used as template for PCR using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ref. 26) (Fig. 1).



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Fig. 1. Representative ethidium bromide-stained agarose gel from a PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers to amplify a 430-bp product. Each group was represented by two replicate pools of 5 animals; "-" indicates negative control (no cDNA added); "M" represents the DNA size marker (1 KB Plus DNA Ladder, GIBCO).

 
Differential RNA Display-PCR
Ten sets of cDNA were synthesized using 10 different anchor primers with the following sequence (5'-3'): T7 promoter-(dT12)-NN, where NN can be AC, GC, CC, AC, AG, GG, CG, AA, GA, or CA. First-strand cDNA synthesis consisted of total RNA (0.2 µg) mixed with one anchor primer (1 µg) and brought to 10 µl with DEPC-treated water. Reactions were as previously described. All cDNA samples were stored at -40°C.

In addition to the ten 3' anchor primers used to generate cDNA, ddPCR utilized 20 different 5' arbitrary primers with the following sequence (5'-3'): M13rev48-random 10-mer (Table 1), resulting in 200 PCR reactions per pool. For all 200 reactions per pool, ddPCR was carried out in the presence of a mix of MgCl2 (3.75 mM), dNTPs (0.25 mM), Taq polymerase (0.5 U), 1x PCR buffer (Sigma-Aldrich, St. Louis, MO), each respective tetramethylrhodamine (TMR) end-labeled 3' anchor primer (0.35 µM), and each respective 5'arbitrary primer (0.35 µM). Conditions for ddPCR reactions were as follows: 95°C for 2 min; 4 cycles 92°C for 15 s, 50°C for 30 s, and 72°C for 2 min; 25 cycles of 92°C for 15 s, 60°C for 30 s, and 72°C for 2 min; followed by 72°C for 7 min and hold at 4°C. PCR reactions were carried out in a Peltier thermal cycler (PTC-220; MJ Research, Waltham, MA)


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Table 1. 3' Arbitrary primers used in ddPCR

 
Four microliters of ddPCR sample product was mixed with 1.5 µl of loading dye (formamide and dextran blue), and the mixture was held at 95°C for 2 min, then placed immediately on ice. Samples were loaded on 5.8% polyacrylamide denaturing gel (Sequagel XR; National Diagnostics, Atlanta, GA) and electrophoresed (3,000 V, 100 W, 55°C) for 5 h in the GenomyxLR DNA sequencer (Genomyx, Foster City, CA). Fluorescent images were captured using the GenomyxSC Fluorescent Imaging Scanner (Genomyx) and analyzed in Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA).

Evaluation of ddPCR products.
Gels were scanned after 2.5 and 5 h of electrophoresis for comparison of changes in gene expression between treatment groups. Amplicons (bands) were scored for significance on the basis of consistency of intensity differences between treatment groups as described by Allan et al. (2). For a gene expression difference to be scored, the two replicates from each harvest group must have been duplicated with the same intensity. Selected bands were excised from the gel using a virtual grid overlay. Gel bands were incubated in a 1.5-ml microcentrifuge tube containing 50 µl of sterile nuclease-free TE solution (10 mM Tris·HCl, pH 7.4) for 1 h at 65°C. Tubes were stored at -80°C until utilized for reamplification.

Reamplification of ddPCR product.
Samples were reamplified for sequencing. The PCR reaction combined 4 µl ddPCR product, MgCl2 (1.5 mM), dNTPs (0.25 mM), Taq polymerase (2 U), 1x PCR buffer (Promega), anchor primer M13 (0.2 µM), and arbitrary primer T7 (0.2 µM) to a total volume of 40 µl. Parameters for PCR were 95°C for 2 min; then 4 cycles of 92°C for 15 s, 50°C for 30 s, 72°C for 2 min; 25 cycles of 92°C for 15 s, 60°C for 30 s, 72°C for 2 min; followed by 72°C for 7 min, and hold at 4°C.

Products from the PCR reamplification reaction were resolved on 2% agarose gels. Bands were excised, and products were purified with a gel purification kit (Qiagen, Santa Clarita, CA). Purified PCR product was sequenced using a DNA sequencer (model 373; Applied Biosystems, Foster City, CA) at the Oklahoma State University DNA Sequencing Core Facility (Stillwater, OK) using M13 universal primer or T7 primer (Promega, Madison, WI). Sequences were screened using the Basic Local Alignment Search Tool (BLAST; Ref. 4) to check for sequence homology with known genes. Reamplified products were ligated into the pCRII plasmid using the TOPO TA Cloning Kit (Invitrogen, San Diego, CA) and resequenced. Following removal of primer sequences, the expressed sequences were submitted to GenBank dbEST (accession numbers listed in Table 3).


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Table 3. Sequence results from ddPCR amplicons

 
Quantification of NAT1 Expression
Structure of NAT1.
Novel APOBEC-1 target-1 (NAT1) is homologous to the carboxyl two-thirds of the eukaryotic initiation factor 4G (eIF4G; Ref. 25). NAT1 was first identified as a substrate for the apolipoprotein B editing catalytic subunit-1 (APOBEC-1), a specific deaminase. This deamination results in specific C-to-U transitions detectable in resulting cDNA as C-T transitions. A region of the 3' end of this mRNA detected by the ddPCR screening was a 98% match for the 3' end of human NAT1 and differed internally by only a single C-T transition from human eIF4G. We used a PCR-based assay to discriminate between these two forms of mRNA expression in adipose samples.

TaqMan quantitative RT-PCR assay.
Target gene expression was evaluated by quantitative RT-PCR utilizing a fluorescent reporter and 5' exonuclease assay system (TaqMan; PE Biosystems, Foster City, CA). Due to its importance in adipogenesis, expression of the peroxisome-proliferator-activated receptor-{gamma} (PPAR{gamma}) was also examined using the same method. Reverse transcription of total RNA and PCR amplification was performed using the TaqMan One-Step RT-PCR Master Mix Reagents Kit, TaqMan fluorescent probe, and sequence detection primers (PE Biosystems). TaqMan probe specific for target was designed to contain a fluorescent 5' reporter dye tetrachloro-6-carboxyfluorescein (TET) and 3' quencher dye [6-carboxy-N,N,N’,N’-tetramethylrhodamine (TAMRA)]. Each one-step RT-PCR reaction (25 µl) contained the following: 2x Master Mix without uracil-N-glycosylase (12.5 µl), 40x MultiScribe and RNase Inhibitor Mix (0.625 µl), target forward primer (200 nM), target reverse primer (200 nM), fluorescent-labeled target probe (200 nM) designed from the mRNA sequence for NAT1 or eIF4G, and total RNA (5 ng). Forward primer and reverse primer for NAT1 and eIF4G were 5'-GAATTTGAATT-3' and 5'-AATTTCAATACTA-3', respectively. Probes were 5'-TET-GTCCCTCTTATTA-TAMRA-3' and 5'-TET-GTCCCTCCTATTA-TAMRA-3' for NAT1 and eIF4G, respectively. The underscored base in the middle of each probe represents the C-T transition point. The PCR amplification was carried out in the ABI Prism 7700 sequence detection system (PE Biosystems). Thermal cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 repetitive cycles of 95°C for 15 s and 60°C for 1 min. As a normalization control for RNA loading, parallel reactions in the same multiwell plate were performed using 18S ribosomal RNA as target (18S ribosomal control kit, PE Biosystems). Reactions were similar to those described previously for target RT-PCR with the exception of 100 nM primer and probe concentrations and 40 pg total RNA used in normalization reactions. Discrimination between the two target sequences was evaluated using synthetic control target sequences and performing the RT-PCR analysis using matched probe and target sequences compared with mismatched probe and target sequences under similar conditions.

Quantification of gene amplification was made following RT-PCR by determining the threshold cycle (CT) number for TET fluorescence within the geometric region of the semi-log plot generated during PCR. Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. The relative quantification of target gene expression across treatments was evaluated using the comparative CT method. The {Delta}CT value was determined by subtracting the ribosomal CT value for each sample from the target CT value of that sample. Calculation of {Delta}{Delta}CT involves using the highest sample {Delta}CT value (sample with the lowest target expression) as an arbitrary constant to subtract from all other {Delta} CT sample values. Fold changes in the relative gene expression of target were then determined by evaluating the expression, 2-{Delta} {Delta}CT.

Linkage mapping for eIF4G.
A single nucleotide polymorphism marker was developed for bovine eIF4G. The human cDNA (GenBank NM_00148) was compared by BLASTN to the GenBank "other EST" database to identify sequences corresponding to the bovine ortholog. Eighteen expressed sequence tags (ESTs) had significant match homologies. One (GenBank AW357755) was used to search the TIGR bovine gene index to identify a tentative consensus (TC) sequence representing the bovine cDNA (TC87087). This TC was compared by BLASTN to the human genome sequence through National Center for Biotechnology Information draft sequence to identify the position and size of human introns. Primers (5'-AACCACCACGCACTCAAA-3'; 5' -GGTTGAGCTAGTTCCGAAATG-3') were developed to match sequence in the putative 17th and 20th exons of the gene, predicted to produce a 776-bp product from human genomic DNA of the four bulls representing the parents of the MARC linkage mapping population (5), producing a product of ~800 bp. PCR products were sequenced, and a polymorphism (G-C) was detected in intron 18 that was informative in three of the four bulls (position 301 of the STS sequence; accession number AF420315). The MARC reference population was genotyped for the G-C polymorphism using the Mass Array MALDI-TOF mass spectrometry system (Sequenom, San Diego, CA). A total of 158 informative meioses were analyzed with all genotypes in the MARC database (9) using CRIMAP v. 2.4 (8).

Statistical Analysis
Growth and carcass data were analyzed with least-squares methods using the General Linear Models procedure of SAS (16). The model included effects of days fed and group. Mean comparisons were made using Duncan’s multiple range test. Results of quantitative RT-PCR were analyzed by one-way ANOVA using the statistical tools package of Excel 97 (Microsoft, Redman, WA).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Growth and Carcass Traits
Animals were stratified by days on the concentrated diet (20, 86, 121, and 146 days), referred to as groups 1–4, respectively. Due to the difficulty of harvesting intramuscular adipose without contamination from adjacent muscle tissue, results from group 1 will not be discussed (carcass data for group 1 is presented in Table 2). There were no significant differences in beginning weight among groups 2–4 (Table 2). Mean end weights, which corresponded with preselected treatment end-point targets, were different (P < 0.05). Average daily gain was also similar (P > 0.05) among groups 2–4.

Ribeye area was smaller (P < 0.05) in treatment group 2 compared with groups 3 and 4. Hot carcass weight, fat thickness, yield grade, and marbling mean average scores were different (P < 0.05) among the groups. Days on the concentrated diet resulted in 6, 67, and 87% of the steers in groups 2, 3, and 4, respectively, grading US Choice (Table 2).

Differential-Display PCR
After visual examination, 300 bands that were differentially expressed were excised from 40 ddPCR gels. Of the 300 products excised, 70 exhibiting the greatest amount of difference between groups 2 vs. 3 and 4 were reamplified and sequenced. Homology to known genes was detected for 40 of the excised products. One of these, NAT1, was selected for further analysis because of its potential for involvement controlling cell development of adipose tissue. In addition, a homology match was identified for ATP-dependant citrate lyase (Table 3). Thirty of the 70 products exhibited no homology to known genes.

Expression of NAT1 and eIF4G
NAT1 mRNA is homologous to the carboxyl two-thirds of the eIF4G (25). A region of the 3' end of this mRNA detected by the ddPCR screening was a 98% match for the 3' end of the human NAT1 mRNA and differed by only a single C-T transition from human eIF4G (Fig. 2). We used a PCR-based assay to discriminate between these two mRNAs in adipose samples. Using synthetic control DNA, we tested the ability of the probes to discriminate the single base difference between the two target mRNAs. When the probe and synthetic target were matches, the resulting {Delta}Rn values (normalized fluorescence) during the PCR amplification rose at a faster rate and plateaued at a higher level than when the probe and target template were mismatched (Fig. 3).



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Fig. 2. Sequence homology of eukaryotic initiation factor 4G (eIF4G) and novel APOBEC-1 target-1 (NAT1). The NAT1 mRNA is homologous to the carboxyl two-thirds of the eIF4G ("EF4G"). NAT1 was first identified as a substrate for the apolipoprotein B editing catalytic subunit-1 (APOBEC-1), a specific deaminase. This deamination results in specific C-to-U transitions detectable in cDNA as C-T transitions. A region of the 3' end of this mRNA detected by the differential-display PCR (ddPCR) screening was a 98% match (107 of 109 nucleotides) for the 3' end of human NAT1 and differed by only a single C-T transition from human eIF4G. Comparison with the human NAT1 sequence revealed that we had identified the C-T transition mapped by Yamanaka et al. (25) at the extreme 3' position in the cDNA.

 


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Fig. 3. Discrimination between eIF4G and NAT1 expression. We used a PCR-based assay to discriminate between expression of these two forms of mRNA in adipose samples. Using synthetic control DNA, we tested the ability of the probes to discriminate the single-base difference between the two target mRNAs. The top line (•) represents amplification of the eIF4G template in the presence of the eIF4G probe; the next line ({blacktriangleup}) represents amplification of the NAT1 template in the presence of the NAT1 probe. When the probe and synthetic target were matches, the resulting {Delta}Rn values (normalized fluorescence) during the PCR amplification rose at a faster rate and plateaued at a higher level than when the probe and target template were mismatched ({blacksquare} = NAT1 template plus eIF4G probe; x = eIF4G template plus NAT1 probe).

 
The expression of NAT1 mRNA was quantified by the comparative CT method shown in Table 4. The result of this calculation is a 2.7-fold difference in expression of NAT1 mRNA between groups 2 and 4. Thus NAT1 mRNA was expressed at higher levels (P < 0.05) in younger, leaner animals compared with older, fatter animals (Fig. 4).


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Table 4. Quantification of NAT1 and eIF4G gene expression using the comparative CT method

 


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Fig. 4. Quantification of eIF4G vs. NAT1 expression. The expression of NAT1 was quantified by the comparative CT method shown in Table 4. The result of this calculation is a 2.7-fold difference in expression of NAT1 between groups 2 and 4. Mean of 2-{Delta}{Delta}CT is shown; means with different superscripts vary (P < 0.05). Thus NAT1 mRNA was expressed at higher levels (P < 0.05) in intramuscular adipose of younger, leaner animals compared with older, fatter animals. Expression of eIF4G mRNA was quantified as described for NAT1 (Table 4). There was no significant difference in expression of eIF4G among the treatment groups.

 
Expression of eIF4G mRNA was quantified as described above for NAT1 (Table 4). There was no significant difference in expression of eIF4G mRNA among the treatment groups (Fig. 4). Likewise, evaluation of PPAR{gamma} mRNA expression (data not shown) revealed no significant difference across treatment groups.

Linkage Mapping of eIF4G
Multipoint analysis positioned the marker on BTA15, at 45.2 cM of the linkage group on the USDA-MARC genetic map (http://www.marc.usda.gov/genome/cattle/cattle.html). This result is consistent with that predicted by comparative mapping based on localizations of eIF4G in mice (MMU7, 51.5 cM) and humans (HSA11; p15-p15). However, eIF4G appears to map slightly telomeric of the 95% confidence interval for a quantitative trait locus (QTL) influencing beef longissimus dorsi tenderness in steers (10).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Different stages of adipocyte development within the longissimus dorsi provide a model for the comparison of gene expression profiles during the marbling process. In the present study, the preselected days on the concentrated diet allowed steers to reach targeted end points for adipose tissue development in the longissimus dorsi. Carcass characteristics were different between groups, which reflected maturity of the longissimus dorsi as well as adipose development; the degree of intramuscular adipose present (marbling score) were different between group 2 vs. groups 3 and 4.

We did not observe changes in expression of PPAR{gamma} mRNA, either by ddPCR or by quantitative PCR, suggesting that we were not identifying very early events in the preadipocyte to adipocyte transition. Even though no known markers of adipocyte differentiation were identified by homology search among the products isolated, several amplicons did follow patterns that would be expected of a developing adipocyte. Enzymes of lipogenesis were identified through ddPCR. ATP-citrate lyase was observed with ddPCR gels to be greater in expression in older, fatter animals (groups 2 and 3) vs. the younger, leaner animals (results not shown). ATP-citrate lyase plays a key role by providing acyl-CoA from glucose to lactate in bovine adipose tissue and is rate limiting only to lipogenesis from lactate (17, 20). As described by Smith et al. (21), ATP-citrate lyase enzyme activity increased over 10-fold in Angus x Hereford and Red Poll steers during the same age period as steers in this trial. The current study supports these previous studies and suggests that intramuscular adipose may have similar temporal patterns of ATP-citrate lyase gene expression as subcutaneous adipose during the feedlot phase.

The beef industry has expended great effort and resources to identify possible gene markers that would identify animals that have a greater propensity to accumulate intramuscular adipose tissue. In accordance to that goal, it was decided to demonstrate whether eIF4G mapped to an area that correlated with genomic regions that have been previously mapped to exhibit differences in cattle that have a greater chance of depositing intramuscular adipose. Even though eIF4G maps slightly telomeric of an increased marbling region, it does raise speculation that with further research and better understanding of the eIF4G/NAT1 interaction that one or both may possibly be a gene marker for a beef palatability characteristic in the future.

The product homologous to the NAT1 is of particular interest because of its apparent function as a translational repressor (25). Greater mRNA expression in leaner animals suggests that NAT1 may function to repress specific genes during early stages of adipose development/lipid filling. Interestingly, NAT1 was first identified as a target for specific deamination of cytidine in the presence of overexpressed APOBEC-1. We identified NAT1 and discriminated between NAT1 and eIF4G on the basis of one of these specific cytidine deamination sites near the 3' terminus of the mRNA. Simple species differences in bovine eIF4G sequence are not likely, since both transcripts are present. At this point, it is not clear whether there was a shift in deaminase activity in the development of this adipose depot. Such a finding would be of great interest in light of recent reports on the regulatory roles of deaminases in cellular differentiation in the context of immune regulation (1215).


    ACKNOWLEDGMENTS
 
We thank Melanie Allen, Larry Burditt, Amy Chastain, Linda Guenther, Kris Novotny, Thea Pratt, and Leanne Wier at Oklahoma State University, as well as Lori Messer at the Animal Science Molecular Genetics laboratory at the University of Nebraska, for excellent technical and administrative assistance. We are especially appreciative of the efforts of Tim Smith and Gary Bennett at USDA-MARC, who collected the data and performed the linkage analyses relative to mapping of bovine eIF4G.

This work was funded by a generous grant from the Oklahoma Beef Industry Council. We also acknowledge the Oklahoma State University Recombinant DNA/ Protein Resource Facility for the synthesis of synthetic oligonucleotides and the sequencing of cloned cDNA.

This manuscript was approved by the Director, Oklahoma Agricultural Experiment Station, Division of Agriculture and Natural Resources, Stillwater, OK, and is published as paper number 13546 of the Journal Series, Nebraska Agricultural Research Division, University of Nebraska, Lincoln, NE.


    FOOTNOTES
 
Address for reprint requests and other correspondence: J. R. Malayer, Physiological Sciences Dept., 264 McElroy Hall, Stillwater, OK 74078-2006 (E-mail: malayer{at}okstate.edu).

10.1152/physiolgenomics.00095.2001.


    References
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Ailhaud G, Grimaldi P, and Negrel R. Cellular and molecular aspects of adipose tissue development. Annu Rev Nutr 12: 207–233, 1992.[ISI][Medline]
  2. Allan MF, Nielson MK, and Pomp D. Gene expression in hypothalamus and brown adipose tissue of mice divergently selected for heat loss. Physiol Genomics 3: 149–156, 2000.[Abstract/Free Full Text]
  3. Altiok S, Xu M, and Spiegelman B. PPAR{gamma} induces cell cycle withdrawal: inhibition of E2F/DP DNA-binding activity via down-regulation of PP2A. Genes Dev 11: 1987–1998, 1997.[Abstract/Free Full Text]
  4. Altschul S, Gish W, Miller W, Myers E, and Lipman D. Basic local alignment search tool. J Mol Biol 215: 403–410, 1990.[ISI][Medline]
  5. Bishop MD, Kappes SM, Keele JW, Stone RT, Sunden SL, Hawkins GA, Toldo SS, Fries R, Grosz MD, and Yoo J. A genetic linkage map for cattle. Genetics 136: 619–639, 1994.[Abstract/Free Full Text]
  6. Chawla A, Schwarz E, Dimaculangan D, and Lazar M. Peroxisome proliferator-activated receptor (PPAR) gamma: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology 135: 798–800, 1994.[Abstract]
  7. Crouse J, Cross H, and Seideman S. Effects of a grass or grain diet on the quality of three beef muscles. J Anim Sci 58: 619–625, 1984.[ISI]
  8. Green P, Falls K, and Crooks S. CRI-Map Documentation (v. 2.4). St. Louis, MO: Washington University School of Medicine, 1990.
  9. Kappes SM, Keele JW, Stone RT, McGraw RA, Sonstegard TS, Smith TP, Lopez-Corrales NL, and Beattie CW. A second-generation linkage map of the bovine genome. Genome Res 7: 235–249, 1997.[Abstract]
  10. Keele JW, Shackelford SD, Kappes SM, Koohmaraie M, and Stone RT. A region on bovine chromosome 15 influences beef longissimus tenderness in steers. J Anim Sci 77: 1364–1371, 1999.[Abstract/Free Full Text]
  11. MacDougald O and Lane M. Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64: 345–373, 1995.[ISI][Medline]
  12. Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, and Honjo T. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553–563, 2000.[ISI][Medline]
  13. Muramatsu M, Sankaranand VS, Anant S, Sugai M, Kinoshita K, Davidson NO, and Honjo T. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J Biol Chem 274: 18470–18476, 1999.[Abstract/Free Full Text]
  14. Muto T, Muramatsu M, Taniwaki M, Kinoshita K, and Honjo T. Isolation, tissue distribution, and chromosomal localization of the human activation-induced cytidine deaminase (AID) gene. Genomics 68: 85–88, 2000.[ISI][Medline]
  15. Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, Catalan N, Forveille M, Dufourcq-Labelouse R, Gennery A, Tezcan I, Ersoy F, Kayserili H, Ugazio AG, Brousse N, Muramatsu M, Notarangelo LD, Kinoshita K, Honjo T, Fischer A, and Durandy A. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102: 565–575, 2000.[ISI][Medline]
  16. SAS Institute. SAS/STAT Software: Changes and Enhancements Through Release 6.11. Cary, NC: SAS Institute, 1996.
  17. Smith S and Crouse J. Relative contributions of acetate, lactate, and glucose to lipogenesis in bovine intramuscular and subcutaneous adipose tissue. J Nutr 114: 792–800, 1984.[ISI][Medline]
  18. Smith S, Lin K, Wilson J, Lunt D, and Cross H. Starvation depresses acylglycerol biosynthesis in bovine subcutaneous but not intramuscular adipose tissue homogenates. Comp Biochem Biophys B 120: 165–174, 1998.
  19. Smith SB, Lunt DK, and Zembayashi M. Intramuscular fat deposition: the physiological process and the potential for its manipulation. In: Plains Nutr Council Spring Conf (publ. no. AREC 00-22). Amarillo, TX: Texas A&M Research and Extension Center, 2000, p. 1–12.
  20. Smith S and Prior R. Evidence for a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine adipose tissue: enzyme and metabolite levels. Arch Biochem Biophys 211: 192–201, 1981.[ISI][Medline]
  21. Smith S, Prior R, Ferrell C, and Mersmann HJ. Interrelationships among diet, age, fat deposition and lipid metabolism in growing steers. J Nutr 114: 153–162, 1984.[ISI][Medline]
  22. Tang QQ and Lane M. Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation. Genes Dev 13: 2231–2241, 1999.[Abstract/Free Full Text]
  23. United States Department of Agriculture. Official United States standards for grades of carcass beef. In: Code of Federal Regulations, 1997, title 7, chapt. 1, pt. 54, p. 102–154.
  24. Wu Z, Bucher N, and Farmer S. Induction of peroxisome proliferator-activated receptor {gamma} during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPß, C/EBP{delta}, and glucocorticoids. Mol Cell Biol 16: 4128–4136, 1996.[Abstract]
  25. Yamanaka S, Poksay K, Arnold K, and Innerarity T. A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme. Genes Dev 11: 321–333, 1997.[Abstract]
  26. Yelich JV, Pomp D, and Geisert RD. Detection of transcripts for retinoic acid receptors, retinol-binding protein, and transforming growth factors during rapid trophoblastic elongation in the porcine conceptus. Biol Reprod 57: 286–294, 1997.[Abstract]