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Cloning and expression of a chicken α-amylase gene1
Gene 192 ( 1997) 261–270
Cloning and expression of a chicken a-amylase gene1 Bernhard F. Benkel a,*, Thuy Nguyen a, Nav Ahluwalia a, Kaarina I. Benkel b, Donal A. Hickey b a Agriculture and Agri-Food Canada, Centre for Food and Animal Research, Ottawa, Ont., K1A 0C6, Canada b Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ont., K1N 6N5, Canada Received 13 September 1996; received in revised form 25 December 1996; accepted 10 January 1997; Received by A. Nakazawa
Abstract We have isolated and sequenced a genomic clone for a pancreatic a-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY ) of 512 aa that is 83% identical to the human pancreatic a-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the a-amylase produced by the yeast cells was identical to that of the native chicken enzyme. © 1997 Elsevier Science B.V. Keywords: Nucleotide sequence; Gene structure; Transgenic yeast
1. Introduction a-Amylases ( EC 3.2.1.1 ) are widely distributed in nature and have been studied in organisms as diverse as microbes, plants and animals. The nt sequences for amy genes have been determined from a variety of species including bacteria (e.g., Palva, 1982), fungi (e.g., Tsukagoshi et al., 1989), insects (e.g., Boer and Hickey, 1986 ), mammals (e.g., Schibler et al., 1982; Nakamura et al., 1984) and plants (e.g., Rogers and Milliman, 1983 ). Thus, amylase is one of a small number of genes for which information on gene sequence and structure is available from a wide range of evolutionarily diverse species. Chicken amylases have been studied previously at the biochemical (Buonocore et al., 1984; Osman, 1982) and allozymic variation levels ( Yardley et al., 1988). In addition, a partial aa sequence corresponding to the 53 * Corresponding author. Tel. +1 613 7591330; fax: +1 613 7591355. 1 Contribution No. 2403 from the Centre for Food and Animal Research. Abbreviations: aa, amino acid(s); amy, gene, cDNA or mRNA coding for AMY; AMY, amylase enzyme; bp, base pair (s); kb, kilobase (s) or 1000 bp; LA-PCR, long and accurate PCR; nt, nucleotide (s); PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; SDS, sodium dodecyl sulfate; SSC ( 1×), 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 78 - 11 19 ( 9 7 ) 00 10 2 -9
N-terminal aa of ostrich pancreatic AMY has been derived (Oosthuizen et al., 1994 ). However, the sequence of an avian amy gene has not been reported to date, and a molecular probe for chicken a-amylase was not previously available. A primary objective of the present study was to extend the molecular data base on amy genes to include an avian amy sequence. In this paper, we describe the cloning and characterization of a chicken a-amylase gene, as well as the expression of a chicken pancreatic amy cDNA in transgenic yeast cells.
2. Materials and methods 2.1. Isolation and characterization of a chicken genomic amylase clone In order to isolate a chicken genomic a-amylase clone, we screened a commercial chicken library with a murine amy cDNA probe. Approx. 150 000 plaques of a library from a White Leghorn chicken in the vector lFIX II (Stratagene) were screened with the 1.2-kb PstI fragment of plasmid pMPa21 (mouse pancreatic amy cDNA; Hagenbuechle et al., 1980 ). The plaque lifts were prepared as previously described (Benton and Davis, 1977). Hybridization was carried out overnight under condi-
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tions of reduced stringency at 37°C in a standard buer system containing 50% formamide (see Benkel and Gavora, 1993 ). Following hybridization, the filters were washed twice for 15 min each in 2×SSC, 0.1% SDS at 42°C, followed by a single wash in 0.5×SSC, 0.1% SDS at 50°C for 30 min. Autoradiograms were exposed at −70°C with intensifying screens. Eighteen positive signals were detected following the first round of library screening. Ten plaques were chosen at random for second screening, and four of these isolates were still positive after a third round of screening. One clone (lA-1) was chosen for large-scale DNA isolation and fragment subcloning in preparation for sequence analysis.
agarose gels containing formaldehyde (Sambrook et al., 1989), transferred to Hybond N+ membranes (Amersham) by the capillary method, and immobilized by UV irradiation. Northern blots were hybridized to oligomer probes as follows: probes were end labelled with 32P using T4 polynucleotide kinase; blots were hybridized overnight in a buer consisting of 6×SSC, 1×Denhardt’s solution, 0.05% Triton X-100, 100 mg/ml tRNA. For high stringency probing, blots were hybridized at 50°C, then washed twice for 10 min each in 6×SSC, 0.5% Triton X-100 at room temperature, followed by a single wash in the same buer at 50°C. For low stringency probing, the same buers were employed, but blots were hybridized at 37°C and processed with a final wash at 37°C.
2.2. Nucleotide sequence analysis 2.4. Southern blot analysis A series of overlapping subclones spanning the amy genomic locus was constructed from the primary isolate lA-1. The bulk of these clones was constructed by inserting restriction fragments of lA-1 into pUC18. However, for the subcloning of the 5∞ end of the locus, for which no convenient restriction enzyme sites were available, we performed long-range PCR on lA-1 DNA. PCR was carried out using the TaKaRa LA-PCR kit ( long and accurate PCR; PanVera), as recommended by the manufacturer, in combination with the primers T7 (5∞-GTAATACGACTCACTATAGGGC-3∞), which primes in the multiple cloning site region of the vector, and Amy-2D (5∞-TTCTTTGGCTTATTCTAATCT-3∞), which anneals to the amy clone near the upstream end of intron C. The LA-PCR product of about 4 kb was subcloned into pCRII ( Invitrogen) for sequencing. The RT-PCR amplified, pancreatic cDNA clone generated for the yeast expression experiment (see Section 2.5 below) was also completely sequenced. Double-stranded DNA was sequenced by the genewalking method using synthetic oligonucleotide primers. Primers were synthesized on an Applied Biosystems 381A synthesizer and deblocked and desalted before use. Sequencing reactions were performed using the dye terminator cycle sequencing kit as described in the instructions supplied by the manufacturer (Applied Biosystems). The extension products were analysed on an Applied Biosystems 373A automated sequencer, and sequence assembly was performed using MicroGenie software by Beckman (Queen and Korn, 1984 ). 2.3. Characterization of the 5∞ non-coding region Total RNA was isolated from the pancreas of an adult chicken of the semicongenic ev3 line of White Leghorn chickens (Crittenden, 1991) by a modification of the method described by Chomczynski and Sacchi (1987 ), as outlined in the product bulletin supplied with the Trizol reagent (BRL). RNA was separated on
High-molecular-weight DNA from a White Leghorn female ( USDA mapping reference line; Crittenden et al., 1993) was provided by L.B. Crittenden (ADOL, East Lansing, MI, USA). DNA was digested with restriction enzymes and separated on agarose gels by standard methods. Transfer to nylon membranes (Hybond N +; Amersham) was carried out by the capillary method, and DNA was fixed to membranes by UV irradiation. Hybridizations were carried out overnight at either 37°C (low stringency) or 42°C (standard stringency) using the buer system indicated above for plaque lift hybridizations. Blots were washed twice in 2×SSC, 0.1% SDS for 15 min at room temperature, followed by a single wash in 1×SSC, 0.1% SDS for 30 min at 50°C for low stringency probing, or 0.2×SSC, 0.1% SDS at 50°C for standard stringency hybridization. Probes were labelled to high specific activity by the random priming method (Feinberg and Vogelstein, 1983 ). Probes used were: (1) an 841-bp ‘intron-specific’ PCR fragment that spans the 678-bp intron C region, and includes 111 bp of the downstream end of exon 2, and 51 bp of the upstream end of exon 3; (2 ) a 6-kb restriction fragment that spans the bulk of the amy genomic coding region, plus about 2 kb of downstream flanking sequences (Fig. 5 ). 2.5. Chicken pancreatic amylase cDNA synthesis Approx. 2 mg of total RNA (isolated as described in Section 2.3) was used as substrate in a reverse transcription (RT ) reaction using the Perkin Elmer RT-PCR kit components according to the instructions supplied by the manufacturer. The RT reaction was primed with oligo(dT ). The oligonucleotide primers used for the PCR stage of the RT-PCR reaction were designed based on sequence information derived from the genomic amy clone. Primer Chamy-Nhe (5∞-ATGCTAGCTCAGTACAATCCCAACACTCAGGCT-3∞) spans the position in the coding region corresponding to the N terminus
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of the mature enzyme, while primer Chamy-Hin ( 5∞-CGAAGCTTATAACTTGGCATCAACGTGAATTG-3∞) spans the stop codon of the amy coding region. The primers were designed to amplify the region in the amy gene encoding the mature a-amylase peptide. In addition, Chamy-Nhe converts the environment of the signal peptidase cleavage site into an NheI site, while Chamy-Hin adds a HindIII site immediately downstream from the stop codon (enzyme recognition sites are underlined ). The modifications introduced by the PCR primers allow the amplified cDNA to be inserted into a yeast shuttle/expression vector (see Section 2.6). The expression cassette portion of the vector encodes a signal sequence that is modelled on the signal peptide of the Drosophila melanogaster amy gene, and has been modified to accept NheI fragments in frame. PCR amplification of the chicken amy cDNA was performed using the LA-PCR kit as described in the manufacturer’s product bulletin. The amplified fragment was subcloned into the vector pCRII (Invitrogen) and sequenced as described above, prior to insertion into the yeast shuttle/expression vector. 2.6. Expression of the chicken amylase cDNA in yeast The chicken amy cDNA fragment was inserted into a modified version of the yeast shuttle vector pBTI-1 (Boehringer Mannheim) containing the transcription promoter and terminator regions of the D. melanogaster a-amylase gene. The D. melanogaster amy promoter is active in Saccharomyces cerevisiae cells that are cultured in media containing glycerol as carbon source ( Hickey et al., 1994). The construction of the shuttle vector, as well as the procedures used for the transformation of the AH22 cir+ strain of S. cerevisiae and selection of transformed cells are described in Hickey et al. (1994 ). Cells transformed with the recombinant shuttle vector containing the chicken amy cDNA insert were grown for 72 h at 30°C in rich liquid medium (yeast extract/peptone) containing 5% glycerol (w/v) as the carbon source. Under these conditions, AMY activity is secreted by the transformed cells into the extracellular medium. Yeast cells transformed with the shuttle vector lacking the chicken amy cDNA insert were used as controls. For the enzyme assays, cells were removed from the medium by centrifugation. Aliquots of extracellular medium were electrophoresed on non-denaturing polyacrylamide gels, and gels were stained for amylase activity as previously described (Benkel and Hickey, 1986 ). To provide samples of amylases representing a variety of electrophoretic mobilities, tissue samples (mouse pancreas or chicken pancreas) or whole flies (D. melanogaster adults) were homogenized in TE pH 7.5, and water-soluble proteins were recovered following centrifugation.
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3. Results 3.1. Characterization of a chicken pancreatic amylase gene Fig. 1 shows the sequence of the chicken a-amylase gene derived from clone lA-1. The coding region of the gene is interrupted by nine introns that vary in length from 86 bp (intron E) to 678 bp (intron C). The transcribed region spans more than 4200 bp, of which exons make up about 1600 bp and intervening sequences account for the remainder, or about 2600 bp. The sequence shown includes portions of the putative transcription promoter and terminator regions. The upstream flanking region contains three separate TATA box motifs within 350 bp of the ATG. Downstream from the coding region, at 104 bp past the translation stop codon, the chicken amy gene carries a single polyadenylation motif (AATAAA). Fig. 1 also includes the sequencing data for the pancreatic cDNA clone, i.e., the protein-encoding portions of the genomic clone and the corresponding stretches in the cDNA were identical. This result identified the genomic locus as a pancreatic chicken amy gene. The cDNA fragment was generated by RT-PCR using the LA-PCR procedure. LA-PCR was chosen for this application because of its roughly tenfold lower error rate in comparison with conventional PCR (Barnes, 1994), in order to ensure the integrity of the amplified sequence. It should be noted, however, that the cDNA was designed for expression of the mature chicken AMY protein ( 1490 bp) in yeast cells. For this reason, sequence comparison of the cDNA and the genomic clones was technically valid only for the region between the primer annealing sites, or 1443 of the 1490 bp. The nt sequence similarity between the chicken amy gene and its mammalian counterparts is high for the protein-encoding regions, i.e., roughly 73% with murine amylases ( Hagenbuechle et al., 1980 ). This conservation of sequence is limited to the coding portions of exons only; there is no obvious homology downstream from the stop codon or upstream from the ATG, except for two short regions, one 5∞ to the ATG and a second immediately upstream from the proximal TATA box (see Section 3.3). 3.2. The chicken a-amylase protein Translation of the chicken amy exon sequences shown in Fig. 1 into aa yields a preprotein of 512 residues. The 15 N-terminal residues form the putative signal sequence of the preprotein, ending in the motif Cys-Trp-Ala. This motif as well as the glutamine residue at the N terminus of the mature peptide are found in a variety of mammalian a-amylases. The predicted mature enzyme sequence shares extens-
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Fig. 2. Alignment of chicken and human a-amylase sequences. The chicken and human pancreatic AMY aa sequences were compared by the align function of MicroGenie software (Queen and Korn, 1984). Dashes are used to indicate identity, a space indicates the location of the signal peptidase cleavage site in the human prepeptide, and an asterisk (*) shows the location of the single aa gap introduced into the human protein to improve the alignment. The two proteins display a high level of sequence similarity, i.e., 415 of 497 residues ( 83.5%) in the mature enzymes are identical, including a continuous stretch of 72 aa.
ive similarity with AMYs from other sources, i.e., about 80% identity with pig pancreatic AMY ( Kluh, 1981) and roughly 55% with D. melanogaster a-amylase (Boer and Hickey, 1986 ). In addition, 47 of the first 53 aa (89%) of the chicken enzyme are identical to the residues derived by peptide sequencing of this region from ostrich AMY (Oosthuizen et al., 1994). Fig. 2 shows chicken AMY aligned with human pancreatic AMY. Overall, the two proteins are 83% identical including a stretch of 72 continuous identical residues. 3.3. Localization of the 5∞ end of the amylase transcript The distance between the TATA box and the translation start codon for pancreatic amy genes in higher eukaryotes is typically very short. In order to localize the 5∞ end of the chicken amy transcript we prepared Northern blots of total RNA isolated from chicken pancreas. Identical filter strips containing pancreatic RNA were probed with a series of oligomers that spanned the region between the ATG and the nearest TATA motif in the upstream flanking region. Fig. 3 shows that hybridization with an oligomer complementary to the region from the ATG to position −17
resulted in a strong signal consisting of a single band at approx. 1750 nt. In contrast, an oligomer complementary to the region between −8 and −27 from the ATG failed to hybridize to the amy mRNA, even under low stringency conditions (data not shown). Three other oligomers that were designed to anneal at greater distances upstream from the ATG (at −49 to −69; −113 to −134; and −300 to −318, respectively) also failed to hybridize to the amy transcript. In addition, a 5∞-RACE (rapid amplification of cDNA ends) experiment yielded a single amplified band which terminated within the coding region of the signal peptide (data not shown). This localized the 5∞ end of the major chicken amy transcript to the region between −20 and −10 from the ATG. Although the sequences immediately upstream from the ATGs in the chicken and the human pancreatic amy genes show little similarity (Fig. 4 ), the size of the interval between the proximal TATA box and the ATG in the chicken locus ( 41 bp) is only 2 nt longer than the equivalent region in the human pancreatic locus AMY2A (Gumucio et al., 1988). Moreover, the transcription start site for the human pancreatic amy gene, at position −17 from the ATG, is within the range
Fig. 1. Nucleotide and predicted aa sequences of chicken a-amylase. Exons and flanking regions of the gene are shown in upper case letters. The predicted aa sequence is presented below the nt sequence of the protein-encoding portions of gene. The sizes in bp refer to the lengths of complete introns, including the ends which are shown in lower case letters in the figure. The three ‘TATA’ motifs, including the putative functional TATA box nearest the ATG, and polyadenylation motif (AATAAA) are underlined. The nt sequence of the pancreatic amy cDNA was identical to the sequence shown for the protein-coding portions of the genomic clone (see Section 3.1 ). The genomic sequence is GenBank accession No. U63411.
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Fig. 3. Northern analysis of pancreatic RNA. Identical filter strips containing chicken pancreatic RNA were probed with synthetic oligomers complementary to the regions indicated in the upper portion of the figure. Oligomers 1 and 2 did not hybridize to the amy transcript, whereas primers 3 and 4 resulted in strong signals. Filters containing 100 mg dots of plasmids with and without a chicken amy insert were used as controls for Northern hybridizations; the positive control dots are shown below the Northern strips.
determined above for the equivalent motif in the chicken gene. The two regions of obvious sequence similarity in the upstream flanking region are the 8 nt immediately 5∞ of the ATG ( 5/8 matches) and the 11 nt upstream from the TATA box (9/11 positions identical, excluding the TATA motif itself ). 3.4. Southern analysis Southern blot analysis was performed in order to address the following questions. First, the Southern banding pattern provides a rough indication of the number of amy loci in the chicken genome. Second,
hybridization with two dierent probes, one specific to an intron in the chicken pancreatic amy locus, and a second probe that included the bulk of the coding region, was carried out to discriminate between pancreatic and potential salivary gland-specific gene copies. Fig. 5B shows a Southern blot of genomic DNA from a White Leghorn chicken probed with an intron-specific PCR fragment of the chicken pancreatic amy gene. Fig. 5C shows the same blot following removal of the first probe and rehybridization with a probe that spans the bulk of the chicken amy coding region (see Fig. 5A). In each case, the pattern of bands predicted from the restriction map of the sequenced locus is visible. In
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Fig. 4. Comparison of upstream flanking sequences for chicken and human pancreatic amylase genes. Alignment of the 77 nt upstream from the chicken ATG and the equivalent region in the human pancreatic amy gene (AMY2A; Gumucio et al., 1988) by the align function of MicroGenie software (Queen and Korn, 1984). Dashes represent identities, whereas an asterisk (*) indicates a gap introduced to improve alignment. The two blocks of relatively high sequence homology are boxed. The stretch of 8 nt immediately upstream from the ATG contains five matches, whereas nine of the eleven positions upstream from the TATA box are identical between the two genes. The transcription start site of the human pancreatic amy gene is marked with an arrow.
addition, a second set of bands corresponding to a second putative pancreatic locus is also present. Preliminary characterization of the proximal end of the primary clone lA-1 has revealed the presence of a small portion of a second amy-related sequence. The orientation of the second locus is opposite to that of the sequenced locus, and the distance between the translation start codons of the two genes is about 2 kb. Although the nt sequence similarity between the two loci is just over 80%, the incomplete locus contains a deletion of about 1 kb stretching from the 3∞ end of exon 1 to the 5∞ portion of exon 3 (this deletion does not aect hybridization with the ‘intron-specific’ probe). It also contains frameshift mutations and stop codons in frame with the ATG (data not shown). Thus, based on our preliminary evidence, this locus appears to be a pseudogene. Presumably, it is this putative pseudogene that is responsible for the second set of amy-specific bands visible on the autoradiograms. Unfortunately, the portion of this locus contained in lA-1 is too small to allow us to predict its associated restriction pattern. The autoradiograms shown are the result of hybridizations performed under standard conditions of stringency. Hybridization with the longer amy probe was also carried out at low stringency in order to detect any sequences in the chicken genome that show a low level of similarity to the pancreatic amy sequence, i.e., salivary amy genes. The conditions used for low-stringency probings were identical to those used previously to isolate amy clones from a fruit fly library with a mouse probe; the nt sequence similarity between the mouse and fly genes is just under 60% ( Benkel et al., 1987). Autoradiograms from low-stringency hybridizations were identical to the standard stringency blots presented in Fig. 5.
3.5. Expression of a chicken amylase cDNA in yeast The chicken amy gene sequence presented in Fig. 1 carries the hallmarks of a complete, expressed locus. However, the amylase multigene family in higher eukaryotes typically displays a complex organization, consisting of multiple copies of pancreas- and, in some species, salivary gland-specific genes, as well as pseudogenes. In order to determine whether the chicken genomic clone that we sequenced encoded a functional enzyme, we prepared a cDNA clone from chicken pancreatic RNA by RT-PCR and inserted the resulting DNA fragment into a yeast expression vector. The nt sequence of the resulting cDNA clone was identical to the protein-encoding portions of the genomic isolate (see Fig. 1). Expression of the chicken amy cDNA in transformed yeast cells resulted in the secretion of active chicken AMY into the extracellular medium. Furthermore, the banding pattern of the enzyme secreted by the yeast cells was identical to that of the enzyme present in pancreas homogenate prepared from the RNA-donor chicken, both with respect to the migration distance of the major band and the presence of at least two other, more negatively charged, minor activity bands ( Fig. 6). This result indicates that all three electrophoretic variants are encoded in the same gene and that the minor forms dier from the major species by posttranslational modifications.
4. Discussion We have isolated and characterized a pancreatic a-amylase clone from the chicken. The chicken amy
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Fig. 5. Southern analysis. (A) Top line: schematic representation of the chicken pancreatic amy gene based on the sequence presented in Fig. 1. This locus represents the complete, expressed gene referred to in the text; the putative pseudogene (not shown) is located to the left in this figure. Solid lines represent upstream and downstream flanking regions, tall rectangles represent exons, and short rectangles represent introns. The dashed line indicates a portion of the downstream flanking region for which nt sequencing is incomplete. The position of the HindIII site within this region has been established by restriction mapping of cloned DNA. The DNA fragments used as probes for Southern hybridization are shown below the diagram of the gene. Probe 1 is an 841-bp PCR fragment that spans intron C. Probe 2 is a BglII restriction fragment of about 6 kb that contains the bulk of the coding region plus several kb of downstream flanking sequence. Restriction enzyme recognition sites are indicated above the schematic: B, BglII; H, HindIII; S, SacI. (B) Autoradiogram of genomic DNA from a White Leghorn chicken probed with the intron-specific probe from pancreatic locus (probe 1 ) at standard stringency. DNA was restricted with: lanes: 1, SacI; 2, BglII; 3, BglII-HindIII. Arrows mark the locations of bands predicted from the sequence of the pancreatic locus depicted in A. Unmarked bands represent restriction fragments derived from a second pancreatic amy locus, presumably the putative pseudogene located roughly 2 kb upstream from the expressed locus. (C ) Autoradiogram of blot shown in B following removal of intron-specific probe and rehybridization with probe 2 at standard stringency. Lane assignments are the same as in B. Arrows mark bands expected based on the schematic of the sequenced locus. Unmarked bands represent restriction fragments derived from the second amy locus. Hybridization with the same probe under low stringency conditions yielded identical results.
Fig. 6. Expression of a chicken amylase cDNA in yeast cells. A native polyacrylamide gel stained for AMY activity: lanes: 1, mouse pancreas homogenate; 2, D. melanogaster Oregon-R strain adult homogenate; 3, chicken pancreas homogenate; 4, extracellular medium from yeast cells transformed with a control vector; 5, extracellular medium from yeast cells transformed with a recombinant vector containing the chicken amy cDNA.
gene contains nine short to medium length introns, resulting in a locus that spans roughly 4 kb. Thus, the chicken gene is intermediate in size between its murine (Schibler et al., 1982) and D. melanogaster (Boer and Hickey, 1986) counterparts which measure about 10 kb and 2.5 kb, respectively. The positions of the introns within the coding regions of the chicken and the human pancreatic amy genes are identical, except for intron B, which is oset by a single nt in the chicken locus in comparison with the corresponding intron in the human gene (Horii et al., 1987 ). The mature AMY encoded by the chicken gene consists of 497 residues, with a predicted signal peptide 15 aa long. The similarity of the mature enzyme with its mammalian counterparts is high, i.e., over 80% aa identity. In addition, the N-terminal 53 residues show 89% identity with the corresponding region in ostrich pancreatic AMY (Oosthuizen et al., 1994).
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AMY is an extracellular enzyme which is often secreted in copious quantities by the cells producing it. Enzyme activity is easily measured by a simple starchiodine stain following electrophoresis on native polyacrylamide gels (Hickey, 1981). The gel assay, which separates proteins primarily on the basis of net charge, is a very sensitive method for discriminating between a-amylases from dierent sources (Benkel and Hickey, 1986; Hickey et al., 1989). In this study, we have exploited the gel assay system to assess the capacity of the sequenced gene to encode functional AMY. The yeast expression system has previously been shown to produce mammalian AMY with a high level of fidelity (e.g., Shiosaki et al., 1990). Transformation of S. cerevisiae cells with an expression vector carrying the chicken amy cDNA resulted in the secretion of AMY into the extracellular medium. Analysis of the enzyme produced by yeast cells revealed an electrophoretic pattern identical to that found for chicken pancreatic homogenates. Thus, the sequenced gene encodes a functional chicken pancreatic AMY. In some higher vertebrate species, a-amylase production is limited primarily to the pancreas (Mocharla et al., 1990 ). In other species, such as the mouse, where both the salivary glands and the pancreas are major sites of AMY production, a dierent set of amy genes is expressed specifically in each of the two tissues (Boulet et al., 1986). Southern analysis of chicken DNA revealed that the chicken genome carries two distinct copies of the amy gene, which have diverged suciently in nt sequence to yield dierent restriction patterns. One locus is a complete and expressed pancreatic amy gene, while the second copy has several of the hallmarks of a pseudogene. Moreover, no putative salivary gland-specific amy loci were found in chicken DNA. This result is consistent with the lack of AMY activity in chicken salivary tissues (Jerrett and Goodge, 1973). It would be of interest to determine whether the organization of the amy region is maintained in other galliform species, or whether some lineages carry two active amy genes. Our interest in chicken a-amylase stems from its key role in the conversion of dietary starch to simple sugars. Modern breeds of chickens have been selected for a high biomass conversion capacity. This selection has been particularly pronounced in modern lines of meat birds which display phenomenal rates of growth. Increased production rates depend on improved levels of feed conversion and feed eciency. Since chicken feed contains a high proportion of starch, selection for high productivity may have favoured individuals with high levels of a-amylase activity. In the mouse, line-specific dierences in pancreatic AMY activity have been shown to correlate with gene copy numbers (Meisler et al., 1986 ). It will be of interest to examine whether amy
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genes have also undergone amplification in high producing, commercial meat chicken lines.
Acknowledgement The authors thank L.B. Crittenden, Avian Disease and Oncology Laboratory, East Lansing, MI, USA, for providing DNA from the White Leghorn line of the USDA chicken mapping reference population for Southern blots, and Ying Fong, Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ont., Canada, for providing technical assistance. This work was supported by an NSERC Operating Grant to D.A.H.
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Hickey, D.A., 1981. Regulation of amylase activity in Drosophila melanogaster: variation in the number of enzyme molecules produced by dierent amylase genotypes. Biochem. Genet. 19, 783–796. Hickey, D.A., Benkel, B.F. and Magoulas, C., 1989. Molecular biology of enzyme adaptations in higher eukaryotes. Genome 31, 272–283. Hickey, D.A., Benkel, K.I., Fong, F. and Benkel, B.F., 1994. A Drosophila gene promoter is subject to glucose repression in yeast cells. Proc. Natl. Acad. Sci. USA 91, 11109–11112. Horii, A., Emi, M., Tomita, N., Nishide, T., Ogawa, M., Mori, T. and Matsubara, K., 1987. Primary structure of human pancreatic a-amylase gene: its comparison with human salivary a-amylase gene. Gene 60, 57–64. Jerrett, S.A. and Goodge, W.R., 1973. Evidence for amylase in avian salivary glands. J. Morphol. 139, 27–46. Kluh, I., 1981. Amino acid sequence of hog pancreatic a-amylase isoenzyme I. FEBS Lett. 136, 231–234. Meisler, M., Antonucci, T.K., Treisman, L.O., Gumucio, D.L. and Samuelson, L.C., 1986. Interstrain variation in amylase gene copy number and mRNA abundance in three mouse tissues. Genetics 113, 713–722. Mocharla, H., Mocharla, R. an Hodes, M.E., 1990. a-Amylase gene transcription in tissues of normal dog. Nucleic Acids Res. 18, 1031–1036. Nakamura, Y., Ogawa, M., Nishide, T., Kosaki, G., Himeno, S. and Matsubara, K., 1984. Sequences for cDNAs for human salivary and pancreatic a-amylases. Gene 28, 263–270. Oosthuizen, V., Naude´, R.J., Oelofsen, W., Muramoto, K. and Kamiya, H., 1994. Ostrich pancreatic a-amylase: kinetic properties,
amino terminal sequence and subsite structure. Int. J. Biochem. 26, 1313–1321. Osman, A.M., 1982. Amylase in chicken intestine and pancreas. Comp. Biochem. Physiol. 73B, 571–574. Palva, I., 1982. Molecular cloning of a-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis. Gene 19, 81–87. Queen, C. and Korn, L.J., 1984. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 12, 581–599. Rogers, J.C. and Milliman, C., 1983. Isolation and sequence analysis of a barley a-amylase cDNA clone. J. Biol. Chem. 258, 8169–8174. Sambrook, J., Fritsch, E. and Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual. CSHL Press, Cold Spring Harbor, NY. Schibler, U., Pittet, A., Young, R.A., Hagenbuechle, O., Tosi, M., Gellman, S. and Wellauer, P.K., 1982. The mouse a-amylase multigene family: sequence organization of members expressed in the pancreas, salivary gland and liver. J. Mol. Biol. 155, 247–266. Shiosaki, K., Takata, K., Omichi, K., Tomita, N., Horii, A., Ogawa, M. and Matsubara, K., 1990. Identification of a novel a-amylase by expression of a newly cloned human amy3 cDNA in yeast. Gene 89, 253–258. Tsukagoshi, N., Furukawa, M., Nagaba, H., Kirita, N., Tsuboi and Udaka, S., 1989. Isolation of a cDNA encoding Aspergillus oryzae Taka-amylase A: evidence for multiple related genes. Gene 84, 319–327. Yardley, D.G., Gapusan, R.A., Jones, J.E. and Hughes, B.L., 1988. The amylase gene-enzyme system of chickens. I. Allozymic and activity variation. Biochem. Genet. 26, 747–755.
Cloning and expression of a chicken a-amylase gene1 Bernhard F. Benkel a,*, Thuy Nguyen a, Nav Ahluwalia a, Kaarina I. Benkel b, Donal A. Hickey b a Agriculture and Agri-Food Canada, Centre for Food and Animal Research, Ottawa, Ont., K1A 0C6, Canada b Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ont., K1N 6N5, Canada Received 13 September 1996; received in revised form 25 December 1996; accepted 10 January 1997; Received by A. Nakazawa
Abstract We have isolated and sequenced a genomic clone for a pancreatic a-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY ) of 512 aa that is 83% identical to the human pancreatic a-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the a-amylase produced by the yeast cells was identical to that of the native chicken enzyme. © 1997 Elsevier Science B.V. Keywords: Nucleotide sequence; Gene structure; Transgenic yeast
1. Introduction a-Amylases ( EC 3.2.1.1 ) are widely distributed in nature and have been studied in organisms as diverse as microbes, plants and animals. The nt sequences for amy genes have been determined from a variety of species including bacteria (e.g., Palva, 1982), fungi (e.g., Tsukagoshi et al., 1989), insects (e.g., Boer and Hickey, 1986 ), mammals (e.g., Schibler et al., 1982; Nakamura et al., 1984) and plants (e.g., Rogers and Milliman, 1983 ). Thus, amylase is one of a small number of genes for which information on gene sequence and structure is available from a wide range of evolutionarily diverse species. Chicken amylases have been studied previously at the biochemical (Buonocore et al., 1984; Osman, 1982) and allozymic variation levels ( Yardley et al., 1988). In addition, a partial aa sequence corresponding to the 53 * Corresponding author. Tel. +1 613 7591330; fax: +1 613 7591355. 1 Contribution No. 2403 from the Centre for Food and Animal Research. Abbreviations: aa, amino acid(s); amy, gene, cDNA or mRNA coding for AMY; AMY, amylase enzyme; bp, base pair (s); kb, kilobase (s) or 1000 bp; LA-PCR, long and accurate PCR; nt, nucleotide (s); PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; SDS, sodium dodecyl sulfate; SSC ( 1×), 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 78 - 11 19 ( 9 7 ) 00 10 2 -9
N-terminal aa of ostrich pancreatic AMY has been derived (Oosthuizen et al., 1994 ). However, the sequence of an avian amy gene has not been reported to date, and a molecular probe for chicken a-amylase was not previously available. A primary objective of the present study was to extend the molecular data base on amy genes to include an avian amy sequence. In this paper, we describe the cloning and characterization of a chicken a-amylase gene, as well as the expression of a chicken pancreatic amy cDNA in transgenic yeast cells.
2. Materials and methods 2.1. Isolation and characterization of a chicken genomic amylase clone In order to isolate a chicken genomic a-amylase clone, we screened a commercial chicken library with a murine amy cDNA probe. Approx. 150 000 plaques of a library from a White Leghorn chicken in the vector lFIX II (Stratagene) were screened with the 1.2-kb PstI fragment of plasmid pMPa21 (mouse pancreatic amy cDNA; Hagenbuechle et al., 1980 ). The plaque lifts were prepared as previously described (Benton and Davis, 1977). Hybridization was carried out overnight under condi-
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tions of reduced stringency at 37°C in a standard buer system containing 50% formamide (see Benkel and Gavora, 1993 ). Following hybridization, the filters were washed twice for 15 min each in 2×SSC, 0.1% SDS at 42°C, followed by a single wash in 0.5×SSC, 0.1% SDS at 50°C for 30 min. Autoradiograms were exposed at −70°C with intensifying screens. Eighteen positive signals were detected following the first round of library screening. Ten plaques were chosen at random for second screening, and four of these isolates were still positive after a third round of screening. One clone (lA-1) was chosen for large-scale DNA isolation and fragment subcloning in preparation for sequence analysis.
agarose gels containing formaldehyde (Sambrook et al., 1989), transferred to Hybond N+ membranes (Amersham) by the capillary method, and immobilized by UV irradiation. Northern blots were hybridized to oligomer probes as follows: probes were end labelled with 32P using T4 polynucleotide kinase; blots were hybridized overnight in a buer consisting of 6×SSC, 1×Denhardt’s solution, 0.05% Triton X-100, 100 mg/ml tRNA. For high stringency probing, blots were hybridized at 50°C, then washed twice for 10 min each in 6×SSC, 0.5% Triton X-100 at room temperature, followed by a single wash in the same buer at 50°C. For low stringency probing, the same buers were employed, but blots were hybridized at 37°C and processed with a final wash at 37°C.
2.2. Nucleotide sequence analysis 2.4. Southern blot analysis A series of overlapping subclones spanning the amy genomic locus was constructed from the primary isolate lA-1. The bulk of these clones was constructed by inserting restriction fragments of lA-1 into pUC18. However, for the subcloning of the 5∞ end of the locus, for which no convenient restriction enzyme sites were available, we performed long-range PCR on lA-1 DNA. PCR was carried out using the TaKaRa LA-PCR kit ( long and accurate PCR; PanVera), as recommended by the manufacturer, in combination with the primers T7 (5∞-GTAATACGACTCACTATAGGGC-3∞), which primes in the multiple cloning site region of the vector, and Amy-2D (5∞-TTCTTTGGCTTATTCTAATCT-3∞), which anneals to the amy clone near the upstream end of intron C. The LA-PCR product of about 4 kb was subcloned into pCRII ( Invitrogen) for sequencing. The RT-PCR amplified, pancreatic cDNA clone generated for the yeast expression experiment (see Section 2.5 below) was also completely sequenced. Double-stranded DNA was sequenced by the genewalking method using synthetic oligonucleotide primers. Primers were synthesized on an Applied Biosystems 381A synthesizer and deblocked and desalted before use. Sequencing reactions were performed using the dye terminator cycle sequencing kit as described in the instructions supplied by the manufacturer (Applied Biosystems). The extension products were analysed on an Applied Biosystems 373A automated sequencer, and sequence assembly was performed using MicroGenie software by Beckman (Queen and Korn, 1984 ). 2.3. Characterization of the 5∞ non-coding region Total RNA was isolated from the pancreas of an adult chicken of the semicongenic ev3 line of White Leghorn chickens (Crittenden, 1991) by a modification of the method described by Chomczynski and Sacchi (1987 ), as outlined in the product bulletin supplied with the Trizol reagent (BRL). RNA was separated on
High-molecular-weight DNA from a White Leghorn female ( USDA mapping reference line; Crittenden et al., 1993) was provided by L.B. Crittenden (ADOL, East Lansing, MI, USA). DNA was digested with restriction enzymes and separated on agarose gels by standard methods. Transfer to nylon membranes (Hybond N +; Amersham) was carried out by the capillary method, and DNA was fixed to membranes by UV irradiation. Hybridizations were carried out overnight at either 37°C (low stringency) or 42°C (standard stringency) using the buer system indicated above for plaque lift hybridizations. Blots were washed twice in 2×SSC, 0.1% SDS for 15 min at room temperature, followed by a single wash in 1×SSC, 0.1% SDS for 30 min at 50°C for low stringency probing, or 0.2×SSC, 0.1% SDS at 50°C for standard stringency hybridization. Probes were labelled to high specific activity by the random priming method (Feinberg and Vogelstein, 1983 ). Probes used were: (1) an 841-bp ‘intron-specific’ PCR fragment that spans the 678-bp intron C region, and includes 111 bp of the downstream end of exon 2, and 51 bp of the upstream end of exon 3; (2 ) a 6-kb restriction fragment that spans the bulk of the amy genomic coding region, plus about 2 kb of downstream flanking sequences (Fig. 5 ). 2.5. Chicken pancreatic amylase cDNA synthesis Approx. 2 mg of total RNA (isolated as described in Section 2.3) was used as substrate in a reverse transcription (RT ) reaction using the Perkin Elmer RT-PCR kit components according to the instructions supplied by the manufacturer. The RT reaction was primed with oligo(dT ). The oligonucleotide primers used for the PCR stage of the RT-PCR reaction were designed based on sequence information derived from the genomic amy clone. Primer Chamy-Nhe (5∞-ATGCTAGCTCAGTACAATCCCAACACTCAGGCT-3∞) spans the position in the coding region corresponding to the N terminus
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of the mature enzyme, while primer Chamy-Hin ( 5∞-CGAAGCTTATAACTTGGCATCAACGTGAATTG-3∞) spans the stop codon of the amy coding region. The primers were designed to amplify the region in the amy gene encoding the mature a-amylase peptide. In addition, Chamy-Nhe converts the environment of the signal peptidase cleavage site into an NheI site, while Chamy-Hin adds a HindIII site immediately downstream from the stop codon (enzyme recognition sites are underlined ). The modifications introduced by the PCR primers allow the amplified cDNA to be inserted into a yeast shuttle/expression vector (see Section 2.6). The expression cassette portion of the vector encodes a signal sequence that is modelled on the signal peptide of the Drosophila melanogaster amy gene, and has been modified to accept NheI fragments in frame. PCR amplification of the chicken amy cDNA was performed using the LA-PCR kit as described in the manufacturer’s product bulletin. The amplified fragment was subcloned into the vector pCRII (Invitrogen) and sequenced as described above, prior to insertion into the yeast shuttle/expression vector. 2.6. Expression of the chicken amylase cDNA in yeast The chicken amy cDNA fragment was inserted into a modified version of the yeast shuttle vector pBTI-1 (Boehringer Mannheim) containing the transcription promoter and terminator regions of the D. melanogaster a-amylase gene. The D. melanogaster amy promoter is active in Saccharomyces cerevisiae cells that are cultured in media containing glycerol as carbon source ( Hickey et al., 1994). The construction of the shuttle vector, as well as the procedures used for the transformation of the AH22 cir+ strain of S. cerevisiae and selection of transformed cells are described in Hickey et al. (1994 ). Cells transformed with the recombinant shuttle vector containing the chicken amy cDNA insert were grown for 72 h at 30°C in rich liquid medium (yeast extract/peptone) containing 5% glycerol (w/v) as the carbon source. Under these conditions, AMY activity is secreted by the transformed cells into the extracellular medium. Yeast cells transformed with the shuttle vector lacking the chicken amy cDNA insert were used as controls. For the enzyme assays, cells were removed from the medium by centrifugation. Aliquots of extracellular medium were electrophoresed on non-denaturing polyacrylamide gels, and gels were stained for amylase activity as previously described (Benkel and Hickey, 1986 ). To provide samples of amylases representing a variety of electrophoretic mobilities, tissue samples (mouse pancreas or chicken pancreas) or whole flies (D. melanogaster adults) were homogenized in TE pH 7.5, and water-soluble proteins were recovered following centrifugation.
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3. Results 3.1. Characterization of a chicken pancreatic amylase gene Fig. 1 shows the sequence of the chicken a-amylase gene derived from clone lA-1. The coding region of the gene is interrupted by nine introns that vary in length from 86 bp (intron E) to 678 bp (intron C). The transcribed region spans more than 4200 bp, of which exons make up about 1600 bp and intervening sequences account for the remainder, or about 2600 bp. The sequence shown includes portions of the putative transcription promoter and terminator regions. The upstream flanking region contains three separate TATA box motifs within 350 bp of the ATG. Downstream from the coding region, at 104 bp past the translation stop codon, the chicken amy gene carries a single polyadenylation motif (AATAAA). Fig. 1 also includes the sequencing data for the pancreatic cDNA clone, i.e., the protein-encoding portions of the genomic clone and the corresponding stretches in the cDNA were identical. This result identified the genomic locus as a pancreatic chicken amy gene. The cDNA fragment was generated by RT-PCR using the LA-PCR procedure. LA-PCR was chosen for this application because of its roughly tenfold lower error rate in comparison with conventional PCR (Barnes, 1994), in order to ensure the integrity of the amplified sequence. It should be noted, however, that the cDNA was designed for expression of the mature chicken AMY protein ( 1490 bp) in yeast cells. For this reason, sequence comparison of the cDNA and the genomic clones was technically valid only for the region between the primer annealing sites, or 1443 of the 1490 bp. The nt sequence similarity between the chicken amy gene and its mammalian counterparts is high for the protein-encoding regions, i.e., roughly 73% with murine amylases ( Hagenbuechle et al., 1980 ). This conservation of sequence is limited to the coding portions of exons only; there is no obvious homology downstream from the stop codon or upstream from the ATG, except for two short regions, one 5∞ to the ATG and a second immediately upstream from the proximal TATA box (see Section 3.3). 3.2. The chicken a-amylase protein Translation of the chicken amy exon sequences shown in Fig. 1 into aa yields a preprotein of 512 residues. The 15 N-terminal residues form the putative signal sequence of the preprotein, ending in the motif Cys-Trp-Ala. This motif as well as the glutamine residue at the N terminus of the mature peptide are found in a variety of mammalian a-amylases. The predicted mature enzyme sequence shares extens-
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Fig. 2. Alignment of chicken and human a-amylase sequences. The chicken and human pancreatic AMY aa sequences were compared by the align function of MicroGenie software (Queen and Korn, 1984). Dashes are used to indicate identity, a space indicates the location of the signal peptidase cleavage site in the human prepeptide, and an asterisk (*) shows the location of the single aa gap introduced into the human protein to improve the alignment. The two proteins display a high level of sequence similarity, i.e., 415 of 497 residues ( 83.5%) in the mature enzymes are identical, including a continuous stretch of 72 aa.
ive similarity with AMYs from other sources, i.e., about 80% identity with pig pancreatic AMY ( Kluh, 1981) and roughly 55% with D. melanogaster a-amylase (Boer and Hickey, 1986 ). In addition, 47 of the first 53 aa (89%) of the chicken enzyme are identical to the residues derived by peptide sequencing of this region from ostrich AMY (Oosthuizen et al., 1994). Fig. 2 shows chicken AMY aligned with human pancreatic AMY. Overall, the two proteins are 83% identical including a stretch of 72 continuous identical residues. 3.3. Localization of the 5∞ end of the amylase transcript The distance between the TATA box and the translation start codon for pancreatic amy genes in higher eukaryotes is typically very short. In order to localize the 5∞ end of the chicken amy transcript we prepared Northern blots of total RNA isolated from chicken pancreas. Identical filter strips containing pancreatic RNA were probed with a series of oligomers that spanned the region between the ATG and the nearest TATA motif in the upstream flanking region. Fig. 3 shows that hybridization with an oligomer complementary to the region from the ATG to position −17
resulted in a strong signal consisting of a single band at approx. 1750 nt. In contrast, an oligomer complementary to the region between −8 and −27 from the ATG failed to hybridize to the amy mRNA, even under low stringency conditions (data not shown). Three other oligomers that were designed to anneal at greater distances upstream from the ATG (at −49 to −69; −113 to −134; and −300 to −318, respectively) also failed to hybridize to the amy transcript. In addition, a 5∞-RACE (rapid amplification of cDNA ends) experiment yielded a single amplified band which terminated within the coding region of the signal peptide (data not shown). This localized the 5∞ end of the major chicken amy transcript to the region between −20 and −10 from the ATG. Although the sequences immediately upstream from the ATGs in the chicken and the human pancreatic amy genes show little similarity (Fig. 4 ), the size of the interval between the proximal TATA box and the ATG in the chicken locus ( 41 bp) is only 2 nt longer than the equivalent region in the human pancreatic locus AMY2A (Gumucio et al., 1988). Moreover, the transcription start site for the human pancreatic amy gene, at position −17 from the ATG, is within the range
Fig. 1. Nucleotide and predicted aa sequences of chicken a-amylase. Exons and flanking regions of the gene are shown in upper case letters. The predicted aa sequence is presented below the nt sequence of the protein-encoding portions of gene. The sizes in bp refer to the lengths of complete introns, including the ends which are shown in lower case letters in the figure. The three ‘TATA’ motifs, including the putative functional TATA box nearest the ATG, and polyadenylation motif (AATAAA) are underlined. The nt sequence of the pancreatic amy cDNA was identical to the sequence shown for the protein-coding portions of the genomic clone (see Section 3.1 ). The genomic sequence is GenBank accession No. U63411.
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Fig. 3. Northern analysis of pancreatic RNA. Identical filter strips containing chicken pancreatic RNA were probed with synthetic oligomers complementary to the regions indicated in the upper portion of the figure. Oligomers 1 and 2 did not hybridize to the amy transcript, whereas primers 3 and 4 resulted in strong signals. Filters containing 100 mg dots of plasmids with and without a chicken amy insert were used as controls for Northern hybridizations; the positive control dots are shown below the Northern strips.
determined above for the equivalent motif in the chicken gene. The two regions of obvious sequence similarity in the upstream flanking region are the 8 nt immediately 5∞ of the ATG ( 5/8 matches) and the 11 nt upstream from the TATA box (9/11 positions identical, excluding the TATA motif itself ). 3.4. Southern analysis Southern blot analysis was performed in order to address the following questions. First, the Southern banding pattern provides a rough indication of the number of amy loci in the chicken genome. Second,
hybridization with two dierent probes, one specific to an intron in the chicken pancreatic amy locus, and a second probe that included the bulk of the coding region, was carried out to discriminate between pancreatic and potential salivary gland-specific gene copies. Fig. 5B shows a Southern blot of genomic DNA from a White Leghorn chicken probed with an intron-specific PCR fragment of the chicken pancreatic amy gene. Fig. 5C shows the same blot following removal of the first probe and rehybridization with a probe that spans the bulk of the chicken amy coding region (see Fig. 5A). In each case, the pattern of bands predicted from the restriction map of the sequenced locus is visible. In
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Fig. 4. Comparison of upstream flanking sequences for chicken and human pancreatic amylase genes. Alignment of the 77 nt upstream from the chicken ATG and the equivalent region in the human pancreatic amy gene (AMY2A; Gumucio et al., 1988) by the align function of MicroGenie software (Queen and Korn, 1984). Dashes represent identities, whereas an asterisk (*) indicates a gap introduced to improve alignment. The two blocks of relatively high sequence homology are boxed. The stretch of 8 nt immediately upstream from the ATG contains five matches, whereas nine of the eleven positions upstream from the TATA box are identical between the two genes. The transcription start site of the human pancreatic amy gene is marked with an arrow.
addition, a second set of bands corresponding to a second putative pancreatic locus is also present. Preliminary characterization of the proximal end of the primary clone lA-1 has revealed the presence of a small portion of a second amy-related sequence. The orientation of the second locus is opposite to that of the sequenced locus, and the distance between the translation start codons of the two genes is about 2 kb. Although the nt sequence similarity between the two loci is just over 80%, the incomplete locus contains a deletion of about 1 kb stretching from the 3∞ end of exon 1 to the 5∞ portion of exon 3 (this deletion does not aect hybridization with the ‘intron-specific’ probe). It also contains frameshift mutations and stop codons in frame with the ATG (data not shown). Thus, based on our preliminary evidence, this locus appears to be a pseudogene. Presumably, it is this putative pseudogene that is responsible for the second set of amy-specific bands visible on the autoradiograms. Unfortunately, the portion of this locus contained in lA-1 is too small to allow us to predict its associated restriction pattern. The autoradiograms shown are the result of hybridizations performed under standard conditions of stringency. Hybridization with the longer amy probe was also carried out at low stringency in order to detect any sequences in the chicken genome that show a low level of similarity to the pancreatic amy sequence, i.e., salivary amy genes. The conditions used for low-stringency probings were identical to those used previously to isolate amy clones from a fruit fly library with a mouse probe; the nt sequence similarity between the mouse and fly genes is just under 60% ( Benkel et al., 1987). Autoradiograms from low-stringency hybridizations were identical to the standard stringency blots presented in Fig. 5.
3.5. Expression of a chicken amylase cDNA in yeast The chicken amy gene sequence presented in Fig. 1 carries the hallmarks of a complete, expressed locus. However, the amylase multigene family in higher eukaryotes typically displays a complex organization, consisting of multiple copies of pancreas- and, in some species, salivary gland-specific genes, as well as pseudogenes. In order to determine whether the chicken genomic clone that we sequenced encoded a functional enzyme, we prepared a cDNA clone from chicken pancreatic RNA by RT-PCR and inserted the resulting DNA fragment into a yeast expression vector. The nt sequence of the resulting cDNA clone was identical to the protein-encoding portions of the genomic isolate (see Fig. 1). Expression of the chicken amy cDNA in transformed yeast cells resulted in the secretion of active chicken AMY into the extracellular medium. Furthermore, the banding pattern of the enzyme secreted by the yeast cells was identical to that of the enzyme present in pancreas homogenate prepared from the RNA-donor chicken, both with respect to the migration distance of the major band and the presence of at least two other, more negatively charged, minor activity bands ( Fig. 6). This result indicates that all three electrophoretic variants are encoded in the same gene and that the minor forms dier from the major species by posttranslational modifications.
4. Discussion We have isolated and characterized a pancreatic a-amylase clone from the chicken. The chicken amy
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Fig. 5. Southern analysis. (A) Top line: schematic representation of the chicken pancreatic amy gene based on the sequence presented in Fig. 1. This locus represents the complete, expressed gene referred to in the text; the putative pseudogene (not shown) is located to the left in this figure. Solid lines represent upstream and downstream flanking regions, tall rectangles represent exons, and short rectangles represent introns. The dashed line indicates a portion of the downstream flanking region for which nt sequencing is incomplete. The position of the HindIII site within this region has been established by restriction mapping of cloned DNA. The DNA fragments used as probes for Southern hybridization are shown below the diagram of the gene. Probe 1 is an 841-bp PCR fragment that spans intron C. Probe 2 is a BglII restriction fragment of about 6 kb that contains the bulk of the coding region plus several kb of downstream flanking sequence. Restriction enzyme recognition sites are indicated above the schematic: B, BglII; H, HindIII; S, SacI. (B) Autoradiogram of genomic DNA from a White Leghorn chicken probed with the intron-specific probe from pancreatic locus (probe 1 ) at standard stringency. DNA was restricted with: lanes: 1, SacI; 2, BglII; 3, BglII-HindIII. Arrows mark the locations of bands predicted from the sequence of the pancreatic locus depicted in A. Unmarked bands represent restriction fragments derived from a second pancreatic amy locus, presumably the putative pseudogene located roughly 2 kb upstream from the expressed locus. (C ) Autoradiogram of blot shown in B following removal of intron-specific probe and rehybridization with probe 2 at standard stringency. Lane assignments are the same as in B. Arrows mark bands expected based on the schematic of the sequenced locus. Unmarked bands represent restriction fragments derived from the second amy locus. Hybridization with the same probe under low stringency conditions yielded identical results.
Fig. 6. Expression of a chicken amylase cDNA in yeast cells. A native polyacrylamide gel stained for AMY activity: lanes: 1, mouse pancreas homogenate; 2, D. melanogaster Oregon-R strain adult homogenate; 3, chicken pancreas homogenate; 4, extracellular medium from yeast cells transformed with a control vector; 5, extracellular medium from yeast cells transformed with a recombinant vector containing the chicken amy cDNA.
gene contains nine short to medium length introns, resulting in a locus that spans roughly 4 kb. Thus, the chicken gene is intermediate in size between its murine (Schibler et al., 1982) and D. melanogaster (Boer and Hickey, 1986) counterparts which measure about 10 kb and 2.5 kb, respectively. The positions of the introns within the coding regions of the chicken and the human pancreatic amy genes are identical, except for intron B, which is oset by a single nt in the chicken locus in comparison with the corresponding intron in the human gene (Horii et al., 1987 ). The mature AMY encoded by the chicken gene consists of 497 residues, with a predicted signal peptide 15 aa long. The similarity of the mature enzyme with its mammalian counterparts is high, i.e., over 80% aa identity. In addition, the N-terminal 53 residues show 89% identity with the corresponding region in ostrich pancreatic AMY (Oosthuizen et al., 1994).
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AMY is an extracellular enzyme which is often secreted in copious quantities by the cells producing it. Enzyme activity is easily measured by a simple starchiodine stain following electrophoresis on native polyacrylamide gels (Hickey, 1981). The gel assay, which separates proteins primarily on the basis of net charge, is a very sensitive method for discriminating between a-amylases from dierent sources (Benkel and Hickey, 1986; Hickey et al., 1989). In this study, we have exploited the gel assay system to assess the capacity of the sequenced gene to encode functional AMY. The yeast expression system has previously been shown to produce mammalian AMY with a high level of fidelity (e.g., Shiosaki et al., 1990). Transformation of S. cerevisiae cells with an expression vector carrying the chicken amy cDNA resulted in the secretion of AMY into the extracellular medium. Analysis of the enzyme produced by yeast cells revealed an electrophoretic pattern identical to that found for chicken pancreatic homogenates. Thus, the sequenced gene encodes a functional chicken pancreatic AMY. In some higher vertebrate species, a-amylase production is limited primarily to the pancreas (Mocharla et al., 1990 ). In other species, such as the mouse, where both the salivary glands and the pancreas are major sites of AMY production, a dierent set of amy genes is expressed specifically in each of the two tissues (Boulet et al., 1986). Southern analysis of chicken DNA revealed that the chicken genome carries two distinct copies of the amy gene, which have diverged suciently in nt sequence to yield dierent restriction patterns. One locus is a complete and expressed pancreatic amy gene, while the second copy has several of the hallmarks of a pseudogene. Moreover, no putative salivary gland-specific amy loci were found in chicken DNA. This result is consistent with the lack of AMY activity in chicken salivary tissues (Jerrett and Goodge, 1973). It would be of interest to determine whether the organization of the amy region is maintained in other galliform species, or whether some lineages carry two active amy genes. Our interest in chicken a-amylase stems from its key role in the conversion of dietary starch to simple sugars. Modern breeds of chickens have been selected for a high biomass conversion capacity. This selection has been particularly pronounced in modern lines of meat birds which display phenomenal rates of growth. Increased production rates depend on improved levels of feed conversion and feed eciency. Since chicken feed contains a high proportion of starch, selection for high productivity may have favoured individuals with high levels of a-amylase activity. In the mouse, line-specific dierences in pancreatic AMY activity have been shown to correlate with gene copy numbers (Meisler et al., 1986 ). It will be of interest to examine whether amy
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genes have also undergone amplification in high producing, commercial meat chicken lines.
Acknowledgement The authors thank L.B. Crittenden, Avian Disease and Oncology Laboratory, East Lansing, MI, USA, for providing DNA from the White Leghorn line of the USDA chicken mapping reference population for Southern blots, and Ying Fong, Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ont., Canada, for providing technical assistance. This work was supported by an NSERC Operating Grant to D.A.H.
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