ISSN 1607�6729, Doklady Biochemistry and Biophysics, 2011, Vol. 439, pp. 199–201. © Pleiades Publishing, Ltd., 2011.Original Russian Text © E.V. Ilnitskaya, V.V. Radchenko, A.M. Kosyreva, T.M. Shuvaeva, V.M. Lipkin, 2011, published in Doklady Akademii Nauk, 2011, Vol. 439, No. 6,pp. 835–837.

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It is known that the synthesis and secretion ofchitinases and mammalian chitinase�like proteinsdramatically increased under pathophysiologicalconditions [1]. In rat olfactory epithelium mucus wefound a new chitinase�like protein (MW ~ 43 kDa),which has not yet been described in the literature andwhich is related to the mouse YM�1 protein. Inaccordance with the localization (epithelium olfac�ctorium), it was named YM�1olf [2]. Partial tryptichydrolysis of the natural protein allowed us to iden�tify peptides, which overlap approximately 30% ofthe presumable amino acid sequence. To determinethe primary structure of the protein, it was necessaryto establish the complete cDNA sequence. This is thefirst paper to report the sequence of full�lengthcDNA of rat YM�1olf (GenBank accessionno. JF781276).

A stable fraction of total RNA was isolated fromthe olfactory tissue of model animals (acute endotox�icosis [3]) using the RNeasy Minikit (Qiagen, UnitedStates). Total double�stranded cDNA (ds�cDNA),synthesized using the MINT�Universal kit (Euro�gene, Russia) served as a template for synthesizingthe full�length YM�1olf cDNA. Earlier, on the basisof the results of direct sequencing of intact nativeYM�1olf protein, we established the amino acidsequence of an 11�aa N�terminal fragment and ana�lyzed the structure of the peptides produced by tryp�tic hydrolysis [2]. Based on these results, we synthe�sized YM�1olfForNcoI and YM�1olfRevBamHIprimers, flanking the coding region of the gene andcontaining the restriction sites NcoI and BamHI at5' ends (table). The amplification product approxi�mately 1150 bp long, which was obtained using this

Cloning and Analysisof a cDNA Sequence Encoding

New Rat Chitinase�Like ProteinE. V. Ilnitskayaa, V. V. Radchenkoa, A. M. Kosyrevab, T. M. Shuvaevaa,

and Corresponding Member of the RAS V. M. Lipkina

Received May 5, 2011

DOI: 10.1134/S1607672911040132

a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry,Russian Academy of Sciences, ul. Miklukho�Maklaya 16/10, Moscow, 117997 Russia

b Research Institute of Human Morphology,Russian Academy of Medical Sciences, ul. Tsyurupy 3, Moscow, 117418 Russia

BIOCHEMISTRY, BIOPHYSICSAND MOLECULAR BIOLOGY

Specific primers used in the study

No. Primer Nucleotide sequence

1 YM�1olfForNcoI (Y1–T8) 5'�ATCCATGGCATATCAGCTGATGTGCTACTATACC�3'

2 YM�1olfRevBamHI (A374–L380) 5'�AAAGGATCCTTAAAGCTCCTCTTTATAAGCCA�3'

3 3'UTR�YM�1olfFor (L341–T347) 5'�CTGGACATGGATGACTTCACTGG�3'

4 5'UTR�YM�1olfRev (H32–F37) 5'�GCAAAGGCATAGATCAAGTGGG�3'

Note: Region encoded by the given primer is indicated parentheses. The recognition sites for restriction endonuclease NcoI and BamHI inthe structure of the primers are underlined. Start and stop codons are shown in bold.

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DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 439 2011

ILNITSKAYA et al.

pair of primers, was cloned into the pGEM�T vector.The clones were analyzed for the presence of theinsertion by PCR using the standard primers comple�mentary to the vector pGEM�T sequence. Thesequence of the insertion in the selected clones wasdetermined using an automatic sequencer. On thebasis of these data, we have completely establishedthe sequence of the cDNA fragment encoding thefull�length YM�1olf protein.

To obtain the sequence of the 5'� and 3'�untrans�lated regions (UTR) of the YM�1olf gene, the total ds�cDNA from rat olfactory mucosa was closed into aring with the aid of T4 DNA ligase and was used astemplate for inverted PCR [4] with specific primerscomplementary to the terminal regions of the codingpart of the gene (3'UTR�YM�1olfFor and 5'UTR�YM�1olfRev; table). The amplification product700 bp long, containing untranslated regions andregions of the coding sequence, was cloned into thepGEM�T vector, and its structure was determined(Fig. 1). The complete nucleotide sequence of matureYM�1olf mRNA was reconstituted by comparing theobtained results with the coding region of the gene.

Figure 2 shows the nucleotide sequence of the full�length YM�1olf cDNA comprising 1545 bp, on whichall regions typical of the mature mRNA are indicated.Taking into account data on the partial amino acidsequence of the protein [2], we assume that thesequence of the precursor of the mature form of ratYM�1olf includes a 21�aa leader peptide (MAKLI�LATGLAILLYAHLGSS) and contains a tyrosine res�idue at the N terminus (Fig. 2). The full�length ratYM�1olf protein contains 380 amino acid residues.

Thus, we were the first to describe and analyze thesequence of full�length cDNA of the YM�1olf protein,which is expressed in rat olfactory mucosa underexperimental endotoxemia.

ACKNOWLEDGMENTS

We are grateful to O.V. Makarova for her invaluablehelp in preparation and discussion of the experimentalapproach used in the study. This study was supportedby the Program of the Presidium of the Russia Acad�emy of Sciences “Molecular and Cellular Biology”(V.M. Lipkin) and the Russian Foundation for BasicResearch (project no. 11�04�00761).

1

2

3

4

5

6

Fig. 1. Nucleotide sequence of the product of cDNA amplification on a circular template containing 5'� and 3'�untranslatedregions. Designations: 1, polylinker sequences of the pGEM�T vector; 2, sequences of 3'UTR�YM�1olfFor and 5'UTR�YM�1olfRev primers; 3, regions of the known sequences encoding C� and N�terminal fragments of the mature rat YM �1olf protein(the stop codon TAA is shown in a larger font); 4, sequences of CDS�1 adapter and PlugOligo�1 adapter primers (Eurogene, Rus�sia) used for the synthesis of total ds�cDNA; 5, sequence encoding the leader peptide (the initiation codon ATG is shown in alarger font); 6, 5 ', and 3'–untranslated sequence of the full�length rat YM�1olf cDNA. The boundaries of the 700�bp fragmentcloned into the pGEM�T vector (\ … \) and the site of linking the ends of ds�cDNA during inverted ligation (…/…) are shown.

DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 439 2011

CLONING AND ANALYSIS OF A cDNA SEQUENCE ENCODING NEW RAT 201

REFERENCES

1. Sutherland, T.E., Maizels, R.M., and Allen, J.E., Clin.Exp. Allergy, 2009, vol. 39, pp. 943–955.

2. Radchenko, V.V., Il’nitskaya, E.V., Tret’yakov, V.E.,et al., Bioorgan.Khimiya, 2010, vol. 36, no. 5, pp. 646–653.

3. Makarova, O.V., Yablonskaya, A.M., Mikhailova, L.P.,et al., Arkh. Patol., 2009, no. 4, pp. 37–43.

4. Ochman, H., Ajioka, J.W., Garza, D., and Hartl, D.L.,Biotechnology, 1990, vol. 18, no. 8, pp. 759–760.

5. Kozak, M., Nucleic Acids Res., 1987, vol. 15, no. 20,pp. 8125–8148.

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Fig. 2. The sequence of the full�length cDNA and the deduced amino acid sequence of the YM�1olf protein, the 5 '�untranslatedregion (highlighted in gray), the start codon ATG (shown in bold italic), the coding region, the stop codon TAA (shown in bolditalic), the 3 '�untranslated region (highlighted in gray), and the polyadenylation site (underlined by the dashed line). The Kozaksequence is framed [5]. The 21�aa detached leader peptide is shown in italic; the regions whose structures were determined earlierexperimentally are underlined [2].

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