Molecular cloning and nucleotide sequence of chicken avidin-related genes 1–5

Eur. J. Biochem. 220, 615-621 (1994) 0 FEBS 1994

Molecular cloning and nucleotide sequence of chicken avidin-related genes 1-5 Riitta A. KEINANEN’, Mika J. WALLEN’, Paula A. KRISTO’, Mikko 0 . LAUKKANEN’, Tarja A. TOIMELA’, Merja A. HELENIUS3 and Markku S. KULOMAA’.’ ‘ University of Tampere, Department of Biomedical Sciences, Finland * Research Laboratory Ltd Alko, Helsinki, Finland

University of Jyvaskyla, Department of Biology, Finland

(Received October 18, 1993) - EJB 93 1564/2

Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1-5, avrI-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88-92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68-78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo

Avidin is a basic glycoprotein found in avian, reptilian and amphibian egg white (Hertz and Sebrell, 1942; Jones and Briggs, 1962; Korpela et al., 1981). It is composed of four identical subunits, each consisting of 128 amino acids, of known sequence (DeLange and Huang, 1971). Avidin is capable of binding the vitamin biotin with an exceptionally tight non-covalent bond (K,, = lo-’’ M ; Green, 1975), suggesting that it may function as an antibiotic protein inhibiting bacterial growth.

Avidin is induced in the oviduct of the estrogen-pre- treated chick by a single steroid hormone, progesterone (O’Malley et al., 1969; Tuohimaa et al., 1989). This progesterone induction requires mRNA synthesis and is followed by a parallel increase in both the avidin mRNA and protein amounts suggesting that the induction occurs mainly at the transcriptional level. In addition, avidin is induced in connection with tissue trauma (Heinonen et al., 1978) and toxic agents (El0 et al., 1975; Heinonen and Tuohimaa, 1978) as well as a consequence of heat injury (Elo, 1980) and bacterial or viral infections (El0 et al., 1980; Korpela et al., 1982; Korpela et al., 1983). This expression is detected in all chicken tissues tested except the brain. Since the inflammation-associated induction needs no estrogen pretreatment and can be abolished by anti-inflammatory drugs (Nordback et al., 1982; Niemela, 1986), the product is called ‘inflainma-

Correspondence to M. S. Kulomaa, University of Jyviiskylii, Department of Biology, P. 0. Box 35, SF-40351 Jyvaskyla, Finland

Fux: +358 41 602 221. Abbreviations. nvr, avidin-related gene ; AVR, avidin-related

protein; PCR, polymerase chain reaction. Note. The nucleotide sequence data reported in this article have

been submitted to the EMBL data bank and are available under the accession numbers 221611, 221554, 221612, 222883 and 222882 for avrl -avr5, respectively. The first two authors contributed equally to this study.

tion avidin’. Altogether, avidin seems to have at least two partially different induction mechanisms (Tuohimaa et al., 1989).

In order to isolate the avidin gene, the cDNA was cloned (Gope et al., 1987). This cDNA was used as a hybridisation probe in the isolation of a genomic clone (AgAV1, previously called llgAV12201; Keinanen et al., 1988). The AgAV1 clone contains three genes closely related to the avidin cDNA. We call these genes avidin-related genes 1 -3, avrl -avr3 (referred to as pgAVl.8, pgAV3.7 and pgAV3.3 in Keinanen et al., 1988), since they are unable to encode the known peptide sequence of egg-white avidin (DeLange and Huang, 1971). In this study, we report molecular cloning of two more members of the chicken avidin gene family and the nucleotide sequence of all five avidin-related genes.

MATERIALS AND METHODS Materials

Restriction and modifying enzymes were purchased from New England Biolabs, Boehringer-Mannheim and Promega. The Sequenase version 2.0 or Sequenase version 1.0 DNA sequencing kit was a product of United States Biochemicals. The AutoRead fluorescent sequencing kit and the Fluore- Prime kit were purchased from Pharmacia. Nylon filter (Hybond-N, 0.45-pm pore size) was obtained from Amer- sham International and [a-”SIdATP, [IX-~’P]~TTP and [a- 12P]dCTP (3000 Ci/mmol) were obtained from NEN Re- search Products. Common laboratory reagents were from Sigma, Merck or J. T. Baker.

Construction and screening of a chicken oviduct genomic DNA library

A chicken genomic library was constructed as previously described (Kleinsek et al., 1986) and was kindly provided by

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Prof. B. W. O'Malley. Briefly, chicken oviduct DNA was partially digested by MboI. 16-24-kb fragments were separated and ligated into the BamHI-digested bacteriophage de- rivative EMBL4 vector before packaging in vitro. Genomic clones were screened using nick translated (Rigby et al., 1977) '2P-labelled chicken avidin cDNA (Gope et al., 1987) probes, either the full-length cDNA or its 3' end (397 bp; positions 198-594), in in situ plaque hybridisation (Benton and Davis, 1977; Keinanen et al., 1988). The Escherichia coli NM539 strain was used to provide host cells.

Characterization of the genomic clones

Recombinant DNA from the genomic clone %gAV2 was isolated for characterization according to Benson and Taylor (1984). The insert was subcloned (Maniatis et al., 1982) into the pBR322 plasmid and an MI3 sequencing vector after Hind111 digestion. Recombinant plasmids were introduced into competent E. coli RR1 cells (Hanahan, 1983).

Plasmid DNA was isolated from the transformants by an alkaline-detergent method (Birnboim and Doly, 1979) and digested with HindIII. After transfer of DNA onto a nylon filter (Southern, 1973, the inserts were characterized by DNA hybridisation analysis. For hybridisations, 20XNaCU Cit was prepared containing 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0. Reagents for SOXDenhardt's solution were 5 g Ficoll (type 400; Pharmacia), 5 g poly(vinylpyrrolidone), 5 g bovine serum albumin (fraction 5 ; Sigma) and H,O to 500 ml. Prehybridisation was performed in 6XNaCl/Cit/ 10XDenhardt's solution/O.l% SDS/SO pglml denatured her- ring sperm DNA at 68°C for at least 4 h with hybridisation overnight at 68°C in an identical solution containing the denatured "P-labelled cDNA probe [full-length probe, or the 5'-end (positions 1 -197), or the 3'-end (positions 198-569) of the cDNA]. Filters were washed at 68°C with 2XNaCII Cit/0.1% SDS (3x5 min and 3X30min) and with 0.1 XNaCl/Cit/O.l% SDS (30 min) before autoradiography. In addition, subclones giving positive hybridisation signals were analysed by restriction enzyme mapping and sequencing.

For genomic blotting, the chromosomal DNA was isolated from chicken muscle (Strauss, 1988) and digested with EcoRI, BamHI or HindIII. DNA was analysed on an 0.8% agarose'gel. The DNA hybridisation analysis was performed as described above using i2P-labelled full-length cDNA as a probe.

Polymerase chain reaction amplification and cloning of the avr3 gene

To obtain the 5' end of the avr3 gene, primers specific for the avr genes and the avidin cDNA (corresponding to nucleotide positions 202-222 and 1302-1321 in Fig. 3) were used to amplify the genes from chicken genomic DNA. As template, 100 ng DNA was used in 50-pl reactions with buffer A (final concentration 50 mM KCI, 10 mM Tris/HCl, pH 9.0, 25"C, with 1.5 mM MgC12, 0.01 % gelatin, 0.1 % Tri- ton X-loo), 50 pmol both primers, 200 pmol of each dNTP and 2.5 U Tuy DNA polymerase overlayed with 100 p1 mineral oil. Amplification reactions took place in a thermal cycler (PTC-100 programmable thermal controller, MJ Re- search). Thirty cycles of amplification (each cycle including denaturation at 94°C for 1 min, primer annealing at 55 "C for 1 min and polymerisation at 72°C for 1.5 min) were performed.

The polymerase-chain-reaction (PCR) products were purified from NuSieve GTG agarose (FMC BioProducts) using Magic PCR Preps (Promega) and cloned using a TA Cloning System version 1.3 (Invitrogen). Plasmid DNA was isolated with Magic Minipreps (Promega). The clones for sequencing were selected by restriction enzyme analysis and large-scale isolation was performed with Magic Maxipreps (Promega).

DNA sequencing The nucleotide sequence was determined by the dideoxy-

nucleotide chain-termination sequencing method (Sanger et al., 1977) using modified T7 DNA polymerase. The Klenow fragment of DNA polymerase I was also used for sequencing of avr l . Double-stranded sequencing of the PCR-generated fragment of avr3 was performed following the instructions of the AutoRead fluorescent sequencing kit (Pharmacia). Analysis of the reaction products was made using a Phar- macia ALF automated DNA sequencer (Ansorge et al., 1987). The primers were either M13 universal or avr-specific primers synthesised with a Cyclone DNA synthesiser (Milli- gen/Biosearch). For automated sequencing, M13 universal primers or avr-specific primers were synthesised with the aid of an Applied Biosystems 381A DNA synthesiser and fluo- rescence-labelled by the Fluoreprime kit from Pharmacia. All sequences were determined from both strands.

Data analysis Sequence data were analysed using programs of the Ge-

netics Computer Group package, version 7.0 (University of Wisconsin Genetics Computer Group, Madison, WI, USA) installed on a Convex C3840 computer (Devereux et al., 1984).

RESULTS Based on the hybridisation analysis of the chicken geno-

mic DNA, a single copy gene for avidin was suggested (Gope et al., 1987). Since three different genes closely related to the avidin cDNA had already been cloned (Keinanen et al., 1988), the hybridisation analysis of genomic DNA was repeated (Fig. 1). Instead of the two bands observed previously (Gope et al., 1987), three bands were detected in the HindIII-digested genomic DNA using the full-length avidin cDNA as a probe. The largest DNA fragment was estimated to be 4.5-5.0 kb. Hybridisation signals obtained from the 3.5-4.0-kb and 2.7-2.9-kb fragments were more intense than signals obtained from the largest fragment, thus suggesting the presence of more than one positive fragment of nearly equal size in both bands. The result confirmed the presence of multiple genes for avidin. In order to isolate the entire avidin gene family, screening of the genomic library was continued.

The second genomic clone, ?,gAV2, was detected after screening of 3.5 X 10'' inore clones from the genomic library (Kleinsek et a]., 1986). By restriction enzyme and hybridisation analyses the two clones were found to be partially overlapping. The insert of AgAV2, approximately 20 kb, was digested with Hind111 and subcloned into pBR322. In addition to avrf-nvr3 (Keinanen et al., 1988), two more members of the gene family, avr4 and avr.5, were isolated.

Since the insert of igAV1 was subcloned as EcoRI fragments (Keinanen et al., 1988), the avr3 gene containing an

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Fig. 1. Hybridisation analysis of chicken genomic DNA. Chromo- somal DNA was isolated from chicken muscle, digested with EcoRI, BarnHI or Hindlll, electrophoresed on an 0.8% agarose gel and hybridised with the full-length avidin cDNA probe as described in the Materials and Methods section (EcoRI, lane 1 ; BarnHI, lane 2; HindIII, lane 3 ) . Molecular-size markers (A/EcoRI, HindIII) are shown (bp).

internal EcoRI site was cut. In order to obtain the 5' end of the gene, PCR was performed. Primers, corresponding to nucleotide positions 202-222 and 1302-1321 in Fig. 3, were used to amplify a fragment of 1.1 kb from the chromosomal DNA, as expected. Based on restriction enzyme analysis of the produced subclones, one of these clones was assumed to contain the region upstream of the EcoRI site of avr3.

Different methods were used to determine the nucleotide sequences of the avr genes. Both strands were sequenced at least once. The length, approximately 1.1 kb, and general structure of the five genes was similar (Fig. 2). Although sequences of the avr4 and uvr5 genes are with a single excep- tion identical, on the basis of restriction enzyme mapping of the HindIII clones (3.9 kb), we consider them to be different genes. All five genes contained four putative exons split by three introns which follow the GT-AG rule at the predicted exon-intron junctions. Identity between the putative exons and the avidin cDNA was 77-98%. The amino acid sequences deduced from the nucleotide sequences of ~ i v r l - avr.5 showed an identity of 68-78% when compared with avidin. Identities of single exons with those of preavidin were 57-100% (Table 1).

DISCUSSION Previous hybridisation studies suggested that avidin was

a single-copy gene (Gope et al., 1987). The nucleotide sequence of the five avidin-related genes (Fig. 3) reveals that all of these cloned genes have a single Hind111 site close to the 3' end. Thus, the identification in this study of three positive bands from the HindIII-digested genomic DNA, two of which are suggested to be at least duplets (Fig. I) , is in keep- ing with current knowledge of the avidin gene family. Hy- bndisations of the genomic DNA were performed using the 3' end (384 bp; Gope et al., 1987) and the full-length cDNA in this study as a probe, but this does not explain the discre-

avr3 FCRCLONB

GEN0MEcsuBCu)NB ECORI

HindlII '1 ECORI avr5 c

d ___) -. A -+ - - t-

Fig. 2. Sequencing strategy for avidin-related genes 1-5. The exons (W) and sequences obtained from the PCR clone containing the 5' end of the avr3 gene ( +) are shown. Sequences from the genomic subclones are also indicated.

- Table 1. Identity between the peptide chain of preavidin and putative avidin-related proteins. The identities are shown for each exon and between the mature avidin (without the signal peptide) and putative avidin-related proteins.

Protein Identity with

exon 1 exon 2 exon 3 exon 4 mature protein

% - ~- ._

AVRl 100 68 73 64 70 AVR2 96 66 73 57 68 AVR3 96 69 78 64 72 AVR4i5 96 69 95 64 78

pancy between the earlier and now reported results. The genomic DNAs were, however, of different origin. In the earlier experiment, DNA was isolated from chicken spleen and in this study DNA was isolated from chicken muscle. It would,

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619

---------- ----_-_-__ - a V K 3

a V K 4

aVK5 _______--_ _ _ _ _ _ _ _ _ _ _ _____----- ---------- - _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _____----- ---------- -

avidin cDNA _ _ _ _ _ _ _ _ _ _ _--_______ ______-___ _--------- -(574) c o n s e n s u s CAGTGCTGAC ACTGCTTGTG ATATTCCTCC CAATAAAGCT T

Fig. 3. Sequence alignment of avidin-related genes 1-5 and avidin cDNA. The hyphens represent the corresponding nucleotide for the consensus sequence; nucleotides that differ from the consensus sequence are shown in small letters. The hyphens on the consensus sequence indicate that no consensus was found. Gaps in the sequence are indicated (.). The actual sequence sizes are 1335 nucleotides (avrl and avr2) due to a gap at nucleotide positions 531 -536 in the figure, 1133 nucleotides (avr3) and 1334 nucleotides (avr4 and avr5), the latter three genes having gaps at positions 531 -536 and position 1011. The sequence of avr3 starts at position 202 and ends at nucleotide 1321. The first and last nucleotides of the cDNA corresponding to putative exons 1-4 in the avr genes are numbered according to the previously published sequence (Cope et al., 1987) in parenthesis.

therefore, be interesting to further study the methylation pattern or other modifications of the corresponding genes in these tissues to see whether they could explain the different results on the complexity of the avidin-encoding nucleotide sequences in the genomic DNA.

The presence of the avr genes in two partially overlapping genomic clones indicates that they lie in the same DNA cluster. The exon-intron junctions of the avr genes agreed well with the general splice junction consensus sequences (Senapathy et al., 1990). Moreover, sequences which are in accordance with the standard branch point consensus sequence were found at expected intron regions, suggesting that the putative transcripts could be properly processed. The presence of a polyadenylation signal (AATAAA) in all genes suggests that the correct modification of the 3' ends could occur. Finally, no internal stop codons were detected in the putative exon sequences. Altogether, this is consistent with the concept that the avr genes are not pseudogenes and could be transcribed into mature mRNAs. In fact, we have shown that at least two of these genes, avr2 and avr3, are transcribed at a low level in the chicken oviduct and intestine, respectively, after intraperitoneal E. coli infection (Kunnas et al., 1993). In addition, avr3 transcripts have also been detected in a chicken macrophage HD-11 cell line (Lappa- lainen, P. J., Kunnas, T. A., Punnonen, E.-L. and Kulomaa, M. S., unpublished results).

Exon 1, encoding the entire signal peptide of the putative avidin-related proteins (AVR) and the first three amino acids of the mature proteins, was the most fully conserved region when the avr genes were compared with the avidin cDNA. Its identity with the cDNA was 97-98%. Exon 2 of all the

avr genes had regions of clustered point mutations, and the identity of the avr genes with the avidin cDNA was 77- 82%. The identity in the third exon was 83-98% and the mutations were again clearly clustered. The fourth exon was also well conserved (92-94%). The point mutations in the introns were more uniformly distributed than in the exons. There was a 6-bp deletion in all avr genes at nucleotide positions 531-536 (exon 2) and a single gap was observed in avr3-avr5 at nucleotide position 1011 (intron 2), but no other deletions or insertions (Fig. 3). The 5' flanking region (up to position - 176 from the predicted transcription-initia- tion site) of a w l , avr2, avr4 and avr5 was perfectly conserved (except for the difference at nucleotide position 10 in Fig. 3), which might predict the expression of the genes, i.e. the promoter region has been subject to evolutionary pres- sure.

The 6-bp deletion in exon 2 would indicate a common ancestor for the avr genes and might be due to 'slipped- strand' mispairing based on the surrounding repeat-like region containing only cytosine and adenine residues. The distribution of point mutations between the avr genes was mark- edly similar, indicating a relatively recent duplication of the ancestral gene. Moreover, avrl and avr2 have many common nucleotides at the sites where they differ from avr4 and avr5, which, as mentioned above, were identical except for the guanine/adenine transition (nucleotide at position 10 in Fig. 3). This would indicate an initial diversion between two genes, one being the ancestor for avrl and avr2. The ancestor for avr4 and avr5, and possibly for uvr3, as indicated by a dendrogram (data not shown) produced by the program PILEUP of the GCG package (Devereux et al., 1984), has

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preAVRl preAVR2 preAVR3 preAVR4/5 preAV SAV


preAVRl preAVR2 preAVR3 preAVR4 /5 preAV SAV


(1 signal peptide 24) 1 26

MVHATSPLLL LLLLSLALVA PgLSARKCSL TGKW IMTIGAVNdn MVHATSPLLL LLLLSLALVA PSLSARKCSL TGeW IMTIGAVNdn MVHtTSPLLL LLLLSLALVA PSLSARKCSL TGKW IMTIrAVNsR MVHtTSPLLL LLLLSLALVA PSLSARKCSL TGKW IMTIrAVNsR MVHATSPLLL LLLLSLALVA PgLSARKCSL TGKW nMTIGAVNsR .......... .......... .... Aaeagi TGtW t f ivtAg . ad 21 GEFnG GN. IT rSPLLGIQH. .K. . RAcQPT FGFTV GEFdG GN.IT LSPLLGIQH. .K..RASQPT FGFTV GEFaG GN . Ik LSPLLGIQH . . K. . RAcQPT FGFTV GEFTG GN.IT LSPLLGIQH. .K..RASQPT FGFTV GEFTG ne.Ik eSPLhGtQnt in..krtQPT FGFTV GaLTG ..aes ryvLtGryds apatdgsgta lGwTV

72 119 VGQCFVDkSG KEVLKT VGQCFVDRSG KEVLKT VGQCFIDRSG KEVLKT tGQCFIDRnG KEVLKT tGQCFIDRnG KEVLKT sGQYv..gga earinT

128 FTRQRTVEE FTRQhTVEE FTRQRTVEE FTRlcTVEE FTRlRTqkE FTkvkpsaa

Fig. 4. Amino acid sequence of the putative avidin-related preproteins (preAVR1-5), preavidin (preAV) and streptavidin (SAV). Amino acids important for the biotin binding of avidin and streptavidin and corresponding amino acids in the UVY genes are shown in boxes. Avidin and streptavidin have been aligned according to Livnah et al. (1 993). The numbering refers to the avidin sequence.

supposedly duplicated more recently than the ancestor for avrl and avr2.

Based on biochemical studies, certain amino acid residues are known to be important for the function of avidin and the bacterial biotin-binding protein streptavidin (Gitlin et al., 1987; Gitlin et al., 1988a; Gitlin et al., 1988b; Kurzban et al., 1989; Weber et al., 1989; Gitlin et al., 1990). Solving the three-dimensional structure of avidin and its functional complex with biotin revealed the amino acid residues responsible for biotin binding (Livnah et al., 1993; Pugliese et a]., 1993). Of the 17 residues, 12-14 are conserved in all AVR species (Fig. 4). Thr38, Ala39 and Thr40, forming hy- drogen bonds with the valeryl moiety of biotin, are substituted in the AVR species. However, these amino acids are also substituted in streptavidin, which folds very similarly with avidin (Weber et al., 1989; Pugliese et al., 1993). Out of the three lysine residues suggested earlier (Gitlin et al., 1987), Lysll l is most likely part of the biotin-binding site (Pugliese et a]., 1993). It is conserved in all AVR species except AVR2. The carbohydrate chain of avidin is attached to Aspl7, which in the putative products of avr genes was substituted by isoleucine. The AVR species, however, contained 2-4 potential glycosylation sites (Asn-Xaa-Thr/ Ser), although the carbohydrate side chain has been suggested to be insignificant for the biotin binding of avidin (Hiller et a]., 1987). Secondary-structure prediction (Chou and Fasman, 1978) of the AVR species showed a relatively

high content of P-sheet structure as predicted for avidin and streptavidin (data not shown). Since the ‘inflammation avidin’ has been detected based on its ability to bind biotin and react with a polyclonal avidin antibody (Tuohimaa et al., 1989), it seems possible that these methods are unable to distinguish avidin from putative avidin-related proteins. Al- together, this suggests that the ‘inflammation avidin’ could be composed of avidin and/or (an) AVRs.

In conclusion, this study indicates that besides the avidin gene, there are five structurally related genes avrl -avr5 in chicken. Avidin is induced in a progesterone-specific manner and in connection with inflammation. Since uvr2 and avr3 transcripts have been detected (Kunnas et al., 1993), it will be interesting to compare how the transcription of avidin and avidin-related genes is regulated. Thus the avidin gene family offers a promising model for the study of the complex mechanisms underlying the reproductive and defense systems.

The authors wish to thank Prof. Bert W. O’Malley (Baylor College of Medicine, Department of Cell Biology, Houston, TX, USA) for providing the chicken genomic library. The skilful techni- cal assistance of Ms Tuula Lehto and Ms Merja Lehtinen is grate- fully acknowledged. Ms Sanna Myohanen, M. Sc. (University of Kuopio, Department of Biochemistry and Biotechnology, Finland) is appreciated for performing the automated DNA sequencing. We thank Dr Timo Ylikomi for critical reading of the manuscript. This study was financially supported by grants from the Rockefeller

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Foundation (New York, NY, USA), the Academy of Finland and the Finnish Cultural Foundation.

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Molecular cloning and nucleotide sequence of chicken avidin-related genes 1–5