Molecular characterization of the 5' regulatory region of rat sodium-dependent multivitamin transporter gene

Nabendu S. Chatterjee1,2, Stanley A. Rubin3,4, and Hamid M. Said1,2

1 Veterans Affairs Medical Center, Long Beach 90822; 2 Departments of Medicine and Physiology/ Biophysics, University of California School of Medicine, Irvine 92697; 3 Veterans Affairs Medical Center, Los Angeles 90073; and 4 Department of Medicine, University of California, Los Angeles, California 90024


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have demonstrated the involvement of a specialized, Na+-dependent carrier-mediated system for biotin uptake in mammalian intestine. The molecular identity of the carrier protein, the Na+-dependent multivitamin transporter (SMVT), has recently been identified. Upon characterization of transcript expression in the rat intestine, four distinct transcript variants (I-IV) due to heterogeneity at the 5'-untranslated region were found (Chatterjee NS, Kumar CK, Ortiz A, Rubin SA, and Said HM. Am J Physiol Cell Physiol 277: C605-C613, 1999). This finding raised the possibility that multiple promoters may be involved in driving the transcription of the SMVT gene. To test this possibility, we cloned the 5' regulatory region of the SMVT gene by genome walking. A 6.5-kb genomic DNA fragment was identified and sequenced. Three putative promoters (P1, P2, and P3) that were separated by exons of the four previously identified transcript variants were, indeed, found. P1 was found to contain multiple putative regulatory regions like GATA-1, AP-1, AP-2, and C/EBP, including several repeats of purine-rich regions and two TATA-like elements. P2 and P3 were GC rich and also revealed the presence of many putative regulatory elements including several SP-1 consensus sequences. The functional identity of each promoter and the minimal regions required for its function were established by the luciferase assay following transfection of rat-derived cultured intestinal epithelial IEC-6 cells. The highest functional activity of the cloned promoters was found to be in the order of P1 > P2 > P3. These findings represent the first characterization of the 5' regulatory region of any mammalian SMVT gene and should assist in the understanding of transcriptional regulation of this important gene.

intestinal biotin transport; gene promoters; transport regulation; multiple variants


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BIOTIN (VITAMIN H) IS AN ESSENTIAL micronutrient that acts as a coenzyme for four carboxylases that catalyze essential steps in four pathways involving fatty acid biosynthesis, gluconeogenesis, and catabolism of certain amino acids and fatty acids (2, 29). The importance of biotin for normal health and well being is underscored by the serious clinical abnormalities that result from its deficiency, which include neurological disorders, growth retardation, and dermal abnormalities (2, 6, 29, 34). In addition, evidence has been recently presented to suggest that marginal biotin deficiency could be teratogenic (34). Biotin deficiency and suboptimal levels are being reported with increased frequency in recent years. Deficiency of biotin occurs in conditions of inborn errors of biotin metabolism, following chronic use of certain anticonvulsant drugs, following long-term use of parenteral nutrition, during pregnancy, in a substantial number of alcoholics, and in patients with inflammatory bowel diseases (6, 10, 12, 29, 33, 34).

Humans and other mammals cannot synthesize biotin, and thus must obtain the vitamin from exogenous sources through absorption in the intestine. Therefore, the intestine plays a central role in determining and regulating normal biotin body homeostasis. Studies over the past 15 years from our laboratory and others have characterized different physiological and biochemical aspects of the intestinal biotin absorption process using a variety of intestinal preparations from different mammalian species (3, 18-27). These studies have shown the involvement of a Na+-dependent, concentrative, carrier-mediated system that is localized at the brush-border membrane of the intestinal absorptive cells. We have also shown that the intestinal biotin uptake process is regulated by dietary biotin deficiency levels (20) and by ontogeny (22). More recently, the intestinal biotin uptake system was also found to be utilized by the unrelated water-soluble vitamin pantothenic acid, and the metabolically important substrate lipoate (18, 23).

The molecular characteristics of the intestinal biotin absorption process of rat, human, and rabbit have been recently delineated by us and others (5, 16). Our studies in rat intestine have identified a cDNA clone that has an open reading frame identical to that of rat placenta, i.e., to that of sodium-dependent multivitamin transporter (SMVT) transcript (15). A significant heterogeneity, however, was found at the 5' untranslated region (5'-UTR) of the rat intestinal transcript with four distinct variants (I to IV) being identified. Furthermore, our findings have also shown the expression of the different variants to be tissue specific in nature (5). For example, variant II was found to be the predominant variant in the small and large intestine. However, this variant was not found in brain, lung, and stomach but was clearly expressed in kidney and liver (5). The finding of multiple variants for the SMVT gene suggests the possible involvement of multiple promoters that drive the expression of this gene. The latter suggestion is based on a substantial number of reports linking heterogeneity at the 5'-UTR of expressed transcripts with existence of multiple promoters (14, 17, 28). Our aim in this study was, therefore, to test this hypothesis and to confirm the existence of the multiple variants at the genomic level. The results obtained have indeed established the existence of multiple (three) promoters in the 5' regulatory region of the SMVT gene and showed that these promoters are separated by exons of the previously identified variants.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. DNA restriction enzymes and Vent DNA polymerase were purchased from New England Biolabs (Beverly, MA). Routine biochemicals were all of molecular biology qualities and were purchased from Fisher Scientific (Tustin, CA). Cell culture reagents were purchased from Sigma Chemical (St. Louis, MO) and Life Technologies (Rockville, MD). Fetal bovine serum was obtained from Omega Scientific (Tarzana, CA). Kits used in this study were purchased from commercial sources as indicated.

Primers. The primers used in different experiments are listed in Table 1. The primers were synthesized by Sigma Genosys (The Woodlands, TX).

                              
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Table 1.   Sequence of primers used in the different PCR reactions

Genome walking. To obtain the genomic DNA sequence of SMVT that includes the promoter, upstream genome walking was performed using Rat GenomeWalker and Advantage Genomic PCR Kits (Clontech, Palo Alto, CA) according to the manufacturer's instructions. The first set of gene-specific primers (primers 1A and 1B, Table 1) was from the vicinity of the ATG initiation codon of the cloned intestinal cDNA. Primary PCR reactions were performed using the flanking adapter-specific primer 1 (AP1, supplied with the kit) and a flanking 3' gene-specific antisense primer, following the manufacturer's protocol and the supplied libraries. A 3.9-kb DNA fragment was obtained from the RDL-1 (EcoRV) library, amplified, and then diluted and used as template for nested secondary PCR using the nested adapter primer (AP2, supplied with the kit) and the respective nested gene-specific antisense primer (primer 1B). The fragment was cloned in pBluescript II SK+ for further characterization. A similar procedure was followed to move further in the upstream direction of the SMVT genomic DNA to obtain the entire 6.5-kb 5' upstream region. In cases where amplification of a genomic fragment proved initially unsuccessful, we suspected a GC-rich template and used a system containing both a reaction buffer and enzyme mix suitable for this purpose (Roche Biochemicals, Indianapolis, IN)

DNA sequence analysis. Subcloned DNA fragments obtained by genomic walking were sequenced with an ABI automated DNA sequencer by a commercial vendor (SeqWright, Houston, TX). Sequence alignment and searches were performed using the BLAST, the TRANSFAC, and the TFSEARCH databases (8, 32).

Primer extension analysis. The transcription start sites of the rat SMVT gene were analyzed by primer extension. Poly(A+) RNA isolated from rat small intestine and colon were isolated as mentioned before (5). Ten picomoles of primers 2R (from exon 1a, variant II), 3R (from exon 1b, variant IV), 4R (from exon 1c, variant I), and 5R (from exon 1d, variant III) (Table 1) were end-labeled with [gamma -32P]ATP and T4 polynucleotide kinase (Promega). The primer extension reactions were carried out with ~8 µg of the poly(A+) RNA, the labeled primers, avian myeloblastosis virus reverse transcriptase, and Primer Extension System (Promega) following manufacturer's procedure. Primers 2R, 3R, and 5R were used with poly(A+) RNA from small intestine, whereas primer 4R was used with poly(A+) RNA from rat colon because this variant (i.e., variant I) was absent in the small intestine (5). The extended product was resolved on SequaGel-8 (National Diagnostics, Atlanta, GA) and visualized by autoradiography. The products of primer extended were compared with an Phi X-174 HinfI DNA marker and 1-bp DNA sequence ladder (not shown).

Promoter-luciferase plasmid construct. Three putative promoter regions (i.e., P1, P2, and P3) were amplified by PCR using specific primers as indicated in Table 2. The PCR fragments were subsequently subcloned upstream of the promoterless luciferase gene in the pGL3-basic vector as described previously (4). The constructs were confirmed by DNA sequencing.

                              
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Table 2.   Preparation of the different promoter and promoter-deletion constructs by PCR

Preparation of different deletion constructs of SMVT promoters. Deletion constructs of the three SMVT promoter P1, P2, and P3 were prepared as follows: the deletion constructs for P1 were prepared using PCR (Table 2). The P1 deletion products were purified and subsequently subcloned into the NheI and HindIII sites of pGL3-basic vector to generate the pGL3-P1Delta constructs. Deletion constructs for P2 and P3 were obtained by suitable restriction digestion to generate pGL3-P2Delta and pGL3-P3Delta constructs, respectively. All constructs were verified with appropriate restriction digestion and DNA sequencing.

Cell cultures and transfection. Promoter-luciferase reporter gene constructs were transfected into rat intestinal epithelial IEC-6 cells (ATCC, Manassas, VA). Approximately 2 × 106 cells were electroporated with 10 µg plasmid that contained the construct as described by us before (5). To normalize for the efficiency of transfection, these cells were also cotransfected with 300 ng of pRL-TK (Promega) plasmid along with the promoter constructs. Total cell lysate was prepared from cells 72 h following transfection, and firefly luciferase activity was measured in these cell extracts using a Turner Design 20/20 Luminometer (Sunnyvale, CA) and was normalized to Renilla luciferase activity in the same cell extract. Protein concentration was measured using Coomassie Plus-200 protein Assay Reagent (Pierce, Rockford, IL). Data presented here are means ± SE of at least three independent experiments and were expressed as relative expression over pGL3-basic, which was set arbitrarily at 1. Statistical analysis was performed using the Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Genomic organization of the 5' regulatory region of rat SMVT gene. Using the genome walking technique described in the MATERIALS AND METHODS, we obtained a 6.5-kb DNA fragment from the 5' regulatory region. The relative position of SMVT gene sequences is based on numbering the "A" of the translation initiation codon sequence as "1" with upstream sequences indicated as negative numbers. This genomic fragment was sequenced and analyzed and was found to contain exons of all the four recently identified transcript variants (i.e., variants I-IV) (Fig. 1). Because these variants possess significant heterogeneity in the extreme 5' end of their UTR, we have named the exons responsible for the heterogeneity as exons 1a to 1d. The common 5'-UTR that exists in all variants was marked as exon 2, the 60-bp placental exon was named exon 2a, and the exon containing the ATG initiation codon was named as exon 3. 


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Fig. 1.   Diagrammatic representation of the 5' regulatory region of the rat Na+-dependent multivitamin transporter (SMVT) gene. The numbers represent nucleotides relative to translation start site (A of ATG is marked as 1). A: the relative positions of the first exons and promoters. Exons are shaded differently. B: the exon composition of the four identified variants of rat SMVT (adapted from figure 1 of Ref . 5). C: the positions of several identified putative regulatory elements. The sequence has been deposited in the GenBank with accession number AF189010.

Our study showed that the region upstream of exon 1a contained multiple putative regulatory regions including several repeats of purine-rich regions and two TATA-like elements. Presence of the several regulatory elements including the TATA-like elements in this region suggests that this region may be a promoter, and this was tentatively named P1. Unlike P1, the region between exons 1a and 1b and that between exons 1b and 1c were found to be GC-rich. Analysis of these regions revealed the presence of many putative regulatory elements, such as SP-1, AP-1, and AP-2. Hence, these areas were also designated as putative promoters P2 and P3, respectively.

Determination of the transcription initiation site. Primer extension analysis for transcription initiation site was carried out using specific primers and poly(A)+ RNA from intestine of rat as described in MATERIALS AND METHODS. The results are shown in Fig. 2. Transcription initiation sites for exon 1a were at -5078, -5133, and -5150; those for exon 1b were at -4426, -4429, and -4442; those for exon 1c were at -4218, -4220, -4225, and -4246; and those for exon 1d were at -4062, -4072, -4115, and -4126. No extended signal was observed with reaction containing no RNA, which was used as a negative control (data not shown).


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Fig. 2.   Determination of the transcription initiation site. Transcription initiation sites are designated by arrows, showing their respective position in the SMVT gene. Lane 1, transcription initiation sites of exon 1a identified by using primer 2R (please see MATERIALS AND METHODS for details); lane 2, transcription initiation sites of exon 1b identified by using primer 3R; lane 3, transcription initiation sites of exon 1c identified by using primer 4R; and lane 4, transcription initiation sites of exon 1d identified by using primer 5R. Lane M indicates the phi X174 marker.

Promoter activity. In an effort to establish the functionality of the three putative promoters (P1, P2, and P3) of the biotin transporter SMVT gene, we prepared three different constructs by cloning these putative promoter regions into the promoterless pGL3-basic vector (Fig. 3) as described previously by Chatterjee et al. (4). The pGL3-basic vector with or without the putative promoter sequences as well as the vector expressing the Renilla luciferase (pRL-TK) were cotransfected into rat intestinal epithelial IEC-6 cells. Activity of the expressed firefly luciferase from the putative promoter constructs and the basic vector was normalized relative to the expressed Renilla luciferase activity. The results showed that transfection of IEC-6 cells with pGL3-P1, pGL3-P2, and pGL3-P3 expressed a 10.5-, 8.6-, and 5.2-fold increase in luciferase activity, respectively, over the promoterless pGL3-basic construct (P < 0.01 for all) (Fig. 3).


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Fig. 3.   Functional analysis of the three identified putative promoters. The size and position of different promoter-luciferase constructs is shown on the left (for details, please see MATERIALS AND METHODS). The results of a luciferase assay of each construct (pGL3-P1, pGL3-P2, and pGL3-P3) following transient transfection into rat intestinal IEC-6 cells are shown on right. Firefly luciferase activity was normalized relative to the activity of simultaneously expressed Renilla luciferase. The results were expressed relative to the pGL3-basic vector, which was arbitrarily set at 1.

To determine the minimum regions required for basal activity of P1, a series of sequentially deleted (Delta ) promoter constructs (Fig. 4) was prepared by PCR (see Table 2 for details). The promoter activity of each construct was then determined by analysis of luciferase expression in the transiently transfected IEC-6 cell extracts. The results showed that (Fig. 4) cells transfected with pGL3-P1Delta 1 have a significant reduction in luciferase activity compared with pGL3-P1. Further deletion of P1 up to -5311 led to a slight reduction in luciferase activity. Upon deletion to -5209, however, a substantial reduction in luciferase activity was observed.


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Fig. 4.   Minimal region required for basal activity of P1. The size and position of different deletion P1-luciferase constructs are shown on the left (for details, please see MATERIALS AND METHODS). The results of a luciferase assay of each construct following transient transfection into rat intestinal IEC-6 cells are shown on the right. Firefly luciferase activity was normalized relative to the activity of simultaneously expressed Renilla luciferase. The results were expressed relative to the pGL3-basic vector, which was set arbitrarily set at 1. *P < 0.01, comparison was made relative to pGL3-P1.

Similarly, different sequentially deleted promoter constructs for P2 were prepared (Fig. 5) by restriction digestion. The results showed that cells transfected with pGL3-P2Delta 1, pGL3-P2Delta 2, pGL3-P2Delta 3, and pGL3-P2Delta 4 displayed a gradually decreased level of luciferase expression compared with pGL3-P2. Further deletions to -4548 (pGL3-P2Delta 5), however, caused an increase in luciferase expression, whereas an additional deletion to -4439 (pGL3-P2Delta 6) led to drastic reduction in luciferase expression.


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Fig. 5.   Minimal region required for basal activity of P2. The size and position of different deletion P2-luciferase constructs are shown on the left (for details, please see MATERIALS AND METHODS). The deletion constructs were prepared by digesting the pGL-P2 with different restriction enzymes that cut once in the insert and once in the multiple cloning sites of the vector followed by religation. The result of a luciferase assay of each construct following transient transfection into rat intestinal IEC-6 cells is shown on the right. Please see legend to Fig. 4, for further description. *P < 0.01 and **P < 0.025, comparison was made relative to pGL3-P2.

To determine the minimum region required for basal activity of P3 promoter, P3Delta constructs (Fig. 6) were transiently transfected into IEC-6 cells following similar methods mentioned earlier. Deletion from -4465 to -4319 led to significant reduction in luciferase expression. Internal deletion from -4441 to -4226 resulted in no luciferase expression of the promoter.


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Fig. 6.   Minimal region required for basal activity of P3. The size and position of different deletion P3-luciferase constructs are shown on the left (for details, please see MATERIALS AND METHODS). The pGL3-P3Delta 1 constructs were prepared by digesting the pGL-P3 with restriction enzymes that cut once in the insert and once in the multiple cloning sites of the vector followed by religation. The pGL3-P3Delta 2 construct was prepared by digesting pGL-P3 with a restriction enzyme (PlmI) that cuts twice in the insert followed by religation. The result of a luciferase assay of each construct following transient transfection into rat intestinal IEC-6 cells is shown on the right. Please see legend to Fig. 4, for further description. *P < 0.01, comparison was made relative to pGL3-P3.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies from our laboratory have identified four unique transcripts characterized as 5'-UTR variants expressed by the biotin transporter SMVT gene (5). These cDNA variants were found to be expressed in a tissue-specific manner (5). The existence of multiple 5'-UTR variants suggested the possible involvement of multiple promoters. To test this possibility, and to begin to understand the regulation of the intestinal biotin uptake process at the molecular level (the intestinal biotin absorption process is known to be regulated by dietary substrate levels and by ontogeny; Refs. 19 and 21), as well as the basis for the tissue-specific expression of the different transcript variants expressed by the SMVT gene, we cloned and characterized the 5' regulatory region of this gene. Our findings provided direct genomic sequence confirmation as the basis for the existence of the different variants, demonstrated their relative position in the upstream region of the biotin transporter SMVT gene, and provided information about their relation to regulatory regions of this gene.

We believe that the beginning of the 5' end of the regulatory region is between -6110 and -6032, as sequence in this upstream region is found to be shared by another rat gene (GenBank accession no. M77850). The 3' end of the regulatory region of the SMVT gene was marked by exon 1d and exon 2. Within the regulatory region, three putative promoters (P1, P2, and P3) were identified that were separated by exons of the four previously identified variants. P1 was found to be 972 bp in length, whereas P2 and P3 were 548 bp and 159 bp in length, respectively. The identification of multiple promoters also suggests the involvement of alternative splicing in the generation of the previously identified distinct SMVT variants (5).

The putative P1 was found to lack any classic TATA or CAAT elements. Instead, two TATA-like elements (4) were found at positions -5193 and -5224 of P1. In addition, P1 was also found to have six putative GATA-1 binding sites. The existence of GATA-1 binding sites in P1 of the SMVT gene is in line with our previous observations of tissue-specific expression of this gene (5), as these sites are known to be involved in tissue-specific expression of other genes (8, 11, 12, 31). In addition to several putative regulatory elements, P1 was found to contain several AP-1 elements and a STAT-binding element, was purine rich, and contained 42% G+C.

As to P2 and P3, these putative promoters were found to be GC rich, containing 66% and 67% G+C, respectively. Again, no classic TATA or CAAT elements were detected at the expected positions in either of these promoters. P2 and P3, however, were found to contain several SP-1, AP-2, and AP-3 elements, which may be involved in basal activity of these promoters (see below). In addition, P2 and P3 were found to contain an AP-1 element, whereas a nuclear factor-kappa B (NF-kappa B) element was found in P2. These putative regulatory elements are known to be inducible under different cellular conditions, e.g., external stimuli (7), but their role in the regulation of SMVT gene transcription is not known and needs further investigation.

The primer extension study suggests that the SMVT gene has multiple transcription initiation sites. From the sequence analysis, it was determined that the transcription initiation sites were located in close vicinity to the proximal TATA-like element in the case of P1 or from the proximal SP-1 elements in case of P2 and P3. The presence of several transcription initiation sites, which were preceded by multiple SP-1 binding sites and the lack of classic TATA or CAAT elements, is a characteristic of a constitutive promoter (1). The physiological significance of having multiple transcription start sites that are close to each other is unclear to us. Similar observations have been reported with other genes that utilize multiple promoters that lack classic TATA or CAAT elements and have multiple transcription start sites (30).

Functional activity of the putative promoter P1 was established by the promoterless luciferase reporter gene expression. Our results showed that among the three putative promoters, P1 was the most active promoter in cultured intestinal epithelial IEC-6 cells; it was followed by P2 and P3, respectively, as shown by the results displayed in Fig. 3. This finding is interesting when related to expression of the different variants in intestinal epithelial cells where variant II was found to be the predominant form (5). This is because the expression of this variant is driven by P1. It is not known at present, however, whether an enhancer is needed for proper functioning of P1 in the intestinal epithelial cells. This issue will be addressed in future investigation.

Our deletion studies to identify the minimum regions needed for basal activity of the different promoters have shown that for P1, a 103-bp sequence from -5311 to -5209 is essential for basal transcription in IEC-6 cells. This region contains a putative TATA-like element along with other regulatory elements including C/EBP, AP-1, and GATA-1. This raises the possibility that these elements may be needed for transcription of the SMVT gene; further studies, however, are needed to test this possibility. The region from -6064 to -5763 was also found to be important for P1 activity expressed in the intestinal epithelial IEC-6 cells.

For P2 and P3, a 110-bp sequence from -4548 to -4439 and a 94-bp sequence from -4441 to -4319, respectively, were found to be needed for basal activity in the intestinal epithelial IEC-6 cells. The minimal promoter region of P2 contains several SP-1 elements and other elements like AP-2, AP-4, Oct-1, and NF-kappa B. The P3 minimal region also contains multiple SP-1 elements. Again, it is not clear whether any of these putative regulatory elements are involved in driving the basal transcription of the SMVT gene in the intestine; further investigations are needed to address these issues. An interesting observation was the finding of a significant increase in P2 activity when a deletion was made from -4600 to -4548. This increase interrupted the progressive decrease in the promoter activity found upon deletion from -4600 to -4548, and suggests possible existence of a negative regulatory element in this region. As stated earlier in this report, variant II is the most predominantly expressed variant in small intestine. The observations that P2 and P3 also show significant activity in IEC-6 cells may indicate the possible existence of a silencer sequence somewhere in the gene.

In summary, results of this study represent the first characterization of the 5' regulatory region of a mammalian biotin transporter gene, SMVT. These findings should serve as a base for future investigation into the molecular regulation of the intestinal biotin absorption process and the pattern of tissue-specific expression of SMVT.


    ACKNOWLEDGEMENTS

The laboratory staff of H. M. Said and S. A. Rubin contributed equally to this work. We thank Scott Smith, Dr. Veedamali S. Subramanian, Alvaro Ortiz, and Ana-Paula E. Duarte for excellent technical help.


    FOOTNOTES

This study was supported by grants from the Department of Veterans Affairs and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56061 and DK-58057.

Address for reprint requests and other correspondence: H. M. Said, VA Medical Center 151, Long Beach, CA 90822 (E-mail: hmsaid{at}uci.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 31 July 2000; accepted in final form 12 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Azizkhan, JC, Jensen DE, Pierce AJ, and Wade M. Transcription from TATA-less promoters: dihydrofolate reductase as a model. Crit Rev Eukaryot Gene Expr 3: 229-254, 1993[Medline].

2.   Bonjour, JP. Biotin. In: Handbook of Vitamins: Nutritional Biochemical and Clinical Aspects, edited by Machlin LJ.. New York: Dekker, 1984, p. 403-435.

3.   Brown, B, Selhub J, and Rosenberg IH. Intestinal absorption of biotin in the rat. J Nutr 116: 1266-1271, 1986[ISI][Medline].

4.   Chatterjee, N, Zou C, Osterman JC, and Gupta NK. Cloning and characterization of the promoter region of a gene encoding a 67-kDa glycoprotein. J Biol Chem 272: 12692-12698, 1997[Abstract/Free Full Text].

5.   Chatterjee, NS, Kumar CK, Ortiz A, Rubin SA, and Said HM. Molecular mechanism of the intestinal biotin transport process. Am J Physiol Cell Physiol 277: C605-C613, 1999[Abstract/Free Full Text].

6.   Dakshinamurti, K, and Chauhan J. Regulation of biotin enzymes. Annu Rev Nutr 8: 211-233, 1988[ISI][Medline].

7.   Faisst, S, and Meyer S. Compilation of vertebrate-encoded transcription factors. Nucleic Acids Res 20: 3-26, 1992[ISI][Medline].

8.   Gregory, RC, Taxman DJ, Seshasayee D, Kensinger MH, Bieker JJ, and Wojchowski DM. Functional interaction of GATA1 with erythroid Kruppel-like factor and Sp1 at defined erythroid promoters. Blood 87: 1793-1801, 1996[Abstract/Free Full Text].

9.   Heinemeyer, T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, and Kolchanov NA. Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26: 362-367, 1998[Abstract/Free Full Text].

10.   Krause, KH, Berlit P, and Bonjour JP. Impaired biotin status in anticonvulsant therapy. Ann Neurol 12: 485-486, 1982[ISI][Medline].

11.   Lemarchandel, V, Ghysdael J, Mignotte V, Rahuel C, and Romeo PH. GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression. Mol Cell Biol 13: 668-676, 1993[Abstract].

12.   Migliaccio, AR, Migliaccio G, Ashihara E, Moroni E, Giglioni B, and Ottolenghi S. Erythroid-specific activation of the distal (testis) promoter of GATA1 during differentiation of purified normal murine hematopoietic stem cells. Acta Haematol 95: 229-235, 1996[ISI][Medline].

13.   Mock, DM, deLorimer AA, Liebman WM, Sweetman L, and Baker H. Biotin deficiency: an unusual complication of parenteral alimentation. N Engl J Med 304: 820-823, 1981[ISI][Medline].

14.   Nakamuta, M, Oka K, Krushkal J, Kobayashi K, Yamamoto M, Li WH, and Chan L. Alternative mRNA splicing and differential promoter utilization determine tissue-specific expression of the apolipoprotein B mRNA editing protein (Apobec) gene in mice. Structure and evaluation of Apobec and related nucleotide deaminases. J Biol Chem 270: 13042-13056, 1995[Abstract/Free Full Text].

15.   Prasad, PD, Wang H, Kekuda R, Fujita T, Feis YJ, Devoe LD, Leibach FH, and Ganapathy V. Cloning and functional expression of a cDNA encoding a mammalian-sodium dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate. J Biol Chem 273: 7501-7506, 1998[Abstract/Free Full Text].

16.   Prasad, PD, Wang H, Huang W, Fei YJ, Leibach FH, Devoe LD, and Ganapathy V. Molecular and functional characterization of the intestinal Na+-dependent multivitamin transporter. Arch Biochem Biophys 366: 95-106, 1999[ISI][Medline].

17.   Rajagopalan, S, Wan DF, Habib GM, Sepulveda AR, McLead MR, Lebovitz RM, and Leiberman MW. Six mRNA with different 5' ends are encoded by a single gamma-glutamyltransferase gene in the mouse. Proc Natl Acad Sci USA 90: 6179-6183, 1993[Abstract].

18.   Said, HM. Cellular uptake of biotin: mechanisms and regulation. J Nutr 129: 490S-493S, 1999[ISI][Medline].

19.   Said, HM, and Derweesh I. A carrier-mediated mechanism for biotin transport in rabbit intestine: studies with brush border membrane vesicles. Am J Physiol Regulatory Integrative Comp Physiol 261: R94-R97, 1991[Abstract/Free Full Text].

20.   Said, HM, Mock DM, and Collins JC. Regulation of intestinal biotin transport in the rat: effect of biotin deficiency and supplementation. Am J Physiol Gastrointest Liver Physiol 256: G306-G311, 1989[Abstract/Free Full Text].

21.   Said, HM, and Mohammedkhani R. Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane. Biochim Biophys Acta 1107: 238-244, 1992[ISI][Medline].

22.   Said, HM, and Redha R. Ontogenesis of the intestinal transport of biotin in the rat. Gastroenterology 94: 68-72, 1988[ISI][Medline].

23.   Said, HM, Ortiz A, McCloud E, Dyer D, Moyer MP, and Rubin S. Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid. Am J Physiol Cell Physiol 275: C1365-C1371, 1998[Abstract/Free Full Text].

24.   Said, HM, and Redha R. Biotin transport in brush border membrane vesicles of rat small intestine. Biochim Biophys Acta 945: 195-201, 1988[ISI][Medline].

25.   Said, HM, Redha R, and Nylander W. A carrier-mediated, Na+ gradient-dependent transport system for biotin in human intestinal brush border membrane vesicles. Am J Physiol Gastrointest Liver Physiol 253: G631-G636, 1987[Abstract/Free Full Text].

26.   Said, HM, Redha R, and Nylander W. Biotin transport in basolateral membrane vesicles of human intestine. Gastroenterology 94: 1157-1163, 1988[ISI][Medline].

27.   Said, HM, Redha R, and Nylander W. Biotin transport in human intestine: site of maximum transport and effect of pH. Gastroenterology 95: 1312-1317, 1988[ISI][Medline].

28.   Schibler, U, and Sierra F. Alternative promoters in developmental gene expression. Annu Rev Genet 21: 237-257, 1987[ISI][Medline].

29.   Sweetman, L, and Nyhan WL. Inheritable biotin-treatable disorders and associated phenomena. Annu Rev Nutr 6: 314-343, 1986.

30.   Tolner, B, Roy K, and Sirotnak FM. Structural analysis of the human RFC-1 gene encoding a folate transporter reveals multiple promoters and alternatively spliced transcripts with 5' end heterogeneity. Gene 211: 331-341, 1998[ISI][Medline].

31.   Tournamille, C, Colin Y, Cartron JP, and Le Van Kim C. Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. Nat Genet 10: 224-228, 1995[ISI][Medline].

32.   Wingender, E, Chen X, Hehl R, Karas H, Liebich I, Matys V, Meinhardt T, Pruss M, Reuter I, and Schacherer F. TRANSFAC: an integrated system for gene expression regulation. Nucleic Acids Res 28: 316-319, 2000[Abstract/Free Full Text].

33.   Wolf, B, Heard GS, Jefferson LG, Proud VK, Nance WI, and Weissbecker KA. Clinical findings in four children with biotinidase deficiency detected through a state-wide neonatal screening program. N Engl J Med 313: 16-19, 1985[Abstract].

34.   Zempleni, J, and Mock DM. Marginal biotin deficiency is teratogenic. Proc Soc Exp Biol Med 223: 14-21, 2000[Abstract/Free Full Text].


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