Biochimica et BiophysicaActa, 1090(1991)273-276 © 1991 ElsevierScience PublishersB.V. All rights reserved0167-4781/91/$03.50 ADONIS 016747819100233T

273

Short Sequence-Paper

BBAEXP 90274

Goose-type lysozyme gene of the chicken: sequence, genomic organization and expression reveals major differences to chicken-type lysozyme gene Toru Nakano * and Thomas Graf Differentiation ~

,

European MolecularBiologyLaboratory, lteidePaerg(Germany)

(Received 13 August 1991)

Key words: Lysozymegene; Nucleicacid sequence;Genomicorganization;(Chicken)

33~s report describes the clonin~ sequencing and expression iuttem of the chicken goose-type lysozyme gene. The cDNA sequence was found to have no bomolosy to that of the chicken.type Jysozyme gene and exhibits a completely different exon-intron organization, in addition, goose-type ly~zyme displays an overlapping but different tissue expression pattern Jn chickens titan chJchen-type lysozyme.

Lysozyme has long been studied as a model for protein structure analyses, molecular evolution and gene regulation [1,9,13,19,23,25,26]. Two distinct forms of lysozyme occur in the egg white of birds. One is typified by the enzyme found in chicken egg white; 'chicken-type' lysozyme [5] and the other by the enzyme found in the egg white of the Embden goose; 'goose.type' lysozyme [6]. Although both types of lysozome display classical muramidase (mucopeptide Nacetylmuramoylhydrolase, EC 3.2.1.17) activity and exhibit similarities in their three-dimensional structure, they differ radically from each other in their amino acid sequence [5,6,9,20,21,22,27]. To our knowledge only one report exists about goose-type lysozyme in chickens. Using specific antibodies this work revealed that chickens contain both types of lysozyme in their ueutrophil 8ranulocytes (hetcrophils) while egg white contains only chick-type lysozyme [11]. In a study where we have screened a eDNA i~rary for chicken granuiocyte-specific sequences regulated by the v-myb oncogene of the avian myeloblastosis virus (AMV) (Nakano

The sequencedata reported in this paper have been submiued to the EMBL/Oenbank Data Libraries under the accession numbers X61001 (senomic)and X61002 (eDNA). * Present address: Department of Medical Chemistry, Faculty of Medicine, KyotoUniversity,Kyoto,Japan. Correspondence: T. Graf, Differentiation Programme, European Molecular BiologyLaboratory,Meyerhofstrasse1, 6900 Heidelberg, Germany.

and Graf, submitted for publication) we have now isolated a clone whose deduced amino acid sequence has a very strong homology with that reported for goose-type lysozyme. Using this clone as a probe we went on to isolate and characterize genomic clones and to determine the expression pattern in chickens of the goose-type lysozyme gene in comparison with that of the chicken-type lysozjme gene. A eDNA i~rary was constructed from poly(A) + m R N A of AMV U°6 v-myb-transformed promyelocytes [12]. To screen the library, labeled cDNA was synthesized from poly(A) + m R N A of AMV Lt°e v-myb-transformed promyelocytes on the one hand or AMV ~ v-myb.transformed monoblasts on the other. By screening a total of 15 000 plaques several clones were identified which hybridized more strongly to the promyelocyte m R N A than to monoblast mRNA (further details are described by Nakano and Graf, submitted for publication). We found that one of the selected clones (clone 325) has a single extended open reading frame of 633 nucleotides, beginning at an A T G codon (position 589) and extending to a T A G termination codon (Fig. 1). The open reading frame potentially encodes a polypeptide of 211 amino acids. The sequence terminates with a poly(A) tail 23 nuclcotides downstream of an A A T A A A polyadenylation signal [18]. The 22 Nterminal amino acids, which are rich in bydmphobic residues, probably function as a signal poptide which is cleaved between two glycines [26] to generate the mature form of the protein. The amino acid sequence predicted from the largest open reading frame of this

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Fig. 1. Nuclnotide sequence of the chicken goose-type b/sozyme gene and its eDNA. Stretches of the genomic seqnence corresponding to the eDNA sequence are indicated in capital letters. The genomlc sequence includes the immediate upstream region. The major transcription initiation site and the polyndenylatinn initiation site are indicated by a closed square and a closed circle, respectively. A 'TATA' box, two 'CAAT' boxes and the polyadenylation signal are underlined, lntrons are positioned according to the terminal GT-AG rule [4]. The sequence of large parts of the intmns are not shown, the nucleotides (nt) missing indicated in the open space. Amino acids of the predicted translation products are shown in a three letter cede. Numherin8 of nucleotides starts with 1 at the transcription initiation site; nmbering of amino acids starts with the first amino acid of the predicted translation products. Numbers are indicated on the right hand side.

Ch~ckan Black Swan Gooao Ost=~ch

.C~¢kan Black Swan Goose Ost=£ch

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ChXcken Black Swan Goos@ Ost:ich

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Ch~e.lum Black Swan Goose Ost::£ch Fig. 2. Comparison of amino acid sequence of chicken S o o s e - ~ lysozyme with that of goose-~ype lysozymes of black swan [22], Embden goose [21] and ostrich [20]. The arrowheads indicate the catalytic center (GIy-T7, Asp.86 and Asp-97) and the closed circles indicate the position of the cysteine residues.

275 clone has a high homology with goose-type lysozyme of black swan [22], Embden goose [21] and ostrich [20] (Fig. 2; percentages of identical amino acids were 81.0, 81.5 and 78.3%, and percentages of similar amino acids were 90.0, 90.2 and 87.0%, respectively). The homology extends over the entire sequence. In addition, both the catalytic center (Glu-73, Asp-86 and Asp-97, Refs. 9 and 27) and all four cysteine residues are conserved. From this strong similarity, we feel confident that clone 325 corresponds to the goose-type lysozome gene of the chicken. Using the eDNA of our chicken goose-type lysozyme gene as a probe, overlapping clones were isolated from a .~ Charon-28 chicken genomic library. Inserts from several independently isolated A phage clones were subeloned into plasmid vectors as described previously [16] and partial restriction maps were determined (data not shown). In total, the overlapping fragments extended over more than 19 kb of genomic DNA, although mapping experiments indicated that the entire gene was contained within a single plasmid clone having a 9.5 kb long insert. The complete nucleotide sequence of the chicken goose-type lysozyme gene contains approx. 3500 bp (Fig. 1). Seouencing of this clone confirmed the sequence of the eDNA clone and identified five introns, their size and exact positions. No nucleotide differences were found between the eDNA and exon sequences. This information and the sequence of 240 bases upstream of the start site is presented in Fig. 1. The major transcription start site (position + 1) was determined by a primer extension assay using a 20-base primer complementary to nucleotides + 72 to + 91 (data not shown). A TATA like sequence (-19 to - 24) and two CAAT sequences (-52 to -55 and -58 to -61) were found in analogy to other promoters [2,3]. No steroid hormone receptor binding sites could be identified in a stretch of about 1.2 kb upstream from the start site (data not shown). However, this does not rule out that such sites exist outside the regions which were sequenced by us. Thus, chicken-type lysozyme contains a progesterone binding site about 2 kb upstream of the transcription initiation site [10]. The three-dimensional structure of goose-type lysozyme exhibits striking similarities to chicken-type lysozyme although it has no or very few similarities in amino acid sequence [9,26]. Weaver et ai. [26] predicted the positions of exon-intron boundaries of goose-type lysozyme on the assumption that exons define structural and function~: traits of both lysozymes [7,14,27]. As shown in Fig. 3, ~he actual exou-intron boundaries of goose-type lysozyme were completely different from the predicted sites. This result demonstrates that the structural similarities between the two lysozymes are not reflected by their genomic organizations.

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Fig. 3. Comparison between goose-type and chicken-type lysozyme genes. Boxes filled with horizontal lines, dots, diagonal lines and vertical lines show 5' nontranslated region, signal peptide region encoding mature lysozyme protein and 3' nontranslated region, respectively. The closed circles indicate the catalytic center (Gly-77, Asp-86 and Asp-97). The solid bars connected by the vertical dashed lines indicate parts of the polypeptide backbones that are similar in their secondary structure between the two types of lysozyme (assuming an identical or highly similar folding, see Ref. 27).

To determine the expression pattern of goose-type lysozyme, RNA was isolated from different tissues of a 4-week-old chick and the RNAs were examined by Northern blotting analysis as described by Ness et al. [16] (Fig. 4). The same filters were stripped and then rehybridized first with chicken-type lysozyme eDNA [24] after which they were stripped again and rehybridized with a/3-actin probe [15] as an internal control. As shown in Fig. 4, the goose-type lysozyme gene was highly expressed in bone marrow and lung, but not

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Fig. 4. Northern blot anal,j~is of chicken goom:-l~pe lystwjqne and chicken-type lysozyme ~ A . I0 ~g of total RNA of bone marrow, spleen, thymus, bursa, liver, intestine, kidney, lung, brain and muscle and 2 /tg of poly(A) + RNA of non-adherent bone marrow cells, adherent bone marrow cells and oviduct were run in denaturing gel containing a 1% formamide and blotted to a nylon filter. The filter was first hybridized with goose-type lysozyme, stripped and rehybridized with chicken-type lysozyme, stripped again and rehybridized with a #-actin probe.

276 detectable in all other organs examined. To analyze the cell type in the bone marrow expressing the gene an adherent fraction consisting essentially of monocytes/ macrophages and a non-adherent fraction consisting of more than 50% promyelocytes (the precursors of granulocytes), was prepared as described earlier [8]. As shown in the same figure, goose-type lysozyme was expressed almost exclusively in the non-adherent population, suggesting that it is specifically expressed in the granulocyte compartment of myelomonocytic cells. This observation confirms the immunological data mentioited earlier [11,17]. In contrast, chicken-type lysozyme was found to be expressed at similar levels in both non-adherent and a~herent populations of bone marrow cells. The high expression of goose-type lysozyme in the lung may be attributable to the presence of either granulocytes or alveolar macrophages. If the latter would be the case, this would mean that the gene is expressed in a specific subpopulation of macrophages. In contrast to chicken-type lysozyme, which is expressed at extremely high levels in the oviduct, expression of goose-type lysozyme was not detectable in this organ. These results indicate that the difference in expression between the two types of lysozyme can be attributed to differences in their transcriptional regulation, and is compatible with the finding that the 1 kb upstream sequences of both genes have no obvious similarities (unpublished observations). Our data cast doubt o n the earlier interpretation based on protein structure comparisons [27] that the two types of avian lysozyme have originated by divergent evolution from a common ancestral gene. Assuming that the two genes have instead arisen by convergent evolution from two different genes it is perhaps all the more surprising that the encoded proteins are not only similar in their three-dimensioual structure but also exhibit overlapping yet distinct patterns of gene expression. Regardless of which interpretation is correct, the role of lysozymes in host defence mechanisms in different tissues must have been the evolutionary pressure which drove the evolution of the two enzymes. We would like to thank C. Bonifer for the oviduct R N A and B. Vennstr6m for the chicken genomic library, S.A. Ness for RNAs, J. Frampton, F. Lim, R. Renkawitz and A. Sippel for comments on this

manuscript. T.N. was supported in part by a fellowship from the Uehara Memorial Foundation. References

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