ISSN 1607�6729, Doklady Biochemistry and Biophysics, 2013, Vol. 452, pp. 241–244. © Pleiades Publishing, Ltd., 2013.Original Russian Text © E.V. Il’nitskaya, V.V. Radchenko, A.S. Rodionova, A.M. Kosyreva, T.M. Shuvaeva, V.M. Lipkin, 2013, published in Doklady Akademii Nauk, 2013,Vol. 452, No. 2, pp. 217–220.

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It is known that the secretion of acid chitinase(AMC�ase, ChiA) in mammals sharply increasesunder pathophysiological conditions. This enzyme isrepresentative of the family of chitinase proteins, theaccumulation of which is a negative prognostic markerof many inflammatory diseases of different etiology[1]. However, the contribution of AMS�ase to thephysiology of these pathologies is not yet understoodcompletely.

This is the first article to describe the detection ofan increased synthesis of two forms of AMC�ase (long,1564 bp in length, and truncated (short, sAMS�ase),1362 bp in length) in an animal model with inflamma�tion induced by a low dose of lipopolysaccharide(LPS). The long form was identified earlier in rodentsand humans under conditions of allergic inflamma�tion [2] (GenBank NM_207586). To determine theprimary structure of the previously unknown shortform of AMC�ase, it was necessary to determine thefull�length sequence of its cDNA. Here, the full�length cDNA sequence of rat sAMS�ase (GenBankKC529649) is presented the first time.

A stable total RNA fraction was isolated from therespiratory lining of model animals with acute endot�oxemia (induced by intraperitoneally injected LPS ata dose of 1.5 mg/kg [3]) using the RNeasy Minikit(Qiagen, United States). The total double�strandedcDNA (ds�cDNA), which was synthesized using theMINT�Universal kit (Evrogen, Russia), served as atemplate for the synthesis of the full�length sAMS�asecDNA. On the basis of the cDNA nucleotide sequenceof the long form of rat AMS�ase, which was taken from

the GenBank database (accession NM_207586),primers 1 and 2 (Table 1), restricting its coding region,were synthesized. With this primer pair, two product1220 and 1422 bp long, corresponding to the cDNA ofthe short and long AMC�ase forms, were amplified(Fig. 1, lane 2). They were excised from the gel, eluted,and cloned into the pGEM�T vector. The analysis ofthese clones for the presence of the insertion was per�formed by PCR using the standard primers comple�

Cloning and Sequence Analysis of cDNA Encoding a New Short Form of Rat Acid Chitinase

E. V. Il’nitskayaa, V. V. Radchenkoa, A. S. Rodionovaa, A .M. Kosyrevab,T. M. Shuvaevaa, and Corresponding Member of the RAS V. M. Lipkina

Received April 18, 2013

DOI: 10.1134/S1607672913050050

a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho�Maklaya 16/10, Moscow, 117997 Russia b Institute of Human Morphology, Russian Academy of Sciences, ul. Tsyurupy 3, Moscow, 117418 Russia

BIOCHEMISTRY, BIOPHYSICSAND MOLECULAR BIOLOGY

1 2 3 54

Fig. 1. Identification of the short form of AMC�ase(sAMC�ase). Electrophoregram of amplification productsof cDNA fragments (25 cycles, 1% agarose gel) containing(1, 2) the coding regions (obtained using primers 1 and 2,see the table) and (4, 5) the untranslated regions (obtainedusing the inverted primers 3 and 4, see the table). The totalcDNA from the respiratory linings of the control (lanes 1, 4)and model (lanes 2, 5) animals was used as a template. 3—Molecular�weight marker SM # 1113 (Fermentas, Lithua�nia).

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mentary to pGEM�T vector sequence. The insertionsof the selected clones were sequenced using a 470Aautomatic gas�phase sequencer (Applied Biosystems470A, United States). On the basis of the obtaineddata, the nucleotide sequence of the cDNA fragmentsencoding the short form of rat AMS�ase (sAMS�ase)and the long form, which was identified earlier (Gen�Bank accession NM_207586), were determined.

The sequences of the 5'� and 3'�untranslatedregions (UTR) of sAMC�ase were determined byinverted PCR [4]. For this purpose, the total ds�cDNAfrom rat respiratory lining was closed in a ring with T4DNA ligase and used as template for PCR performedwith specific primers 3 and 4 (table), which were com�plementary to the terminal regions of the coding partof cDNA. The amplification products 577 and 375 bplong, containing the untranslated regions and the cod�ing sequence regions of the long and short forms ofAMC�ase, respectively (Fig. 1, lane 5), were clonedinto the pGEM�T vector, after which the structure ofthe short form was determined (Fig. 2). As a result of

analysis of the 577�bp fragment sequence and thesAMC�ase coding region, the nucleotide sequence ofthe mature mRNA of the short form of AMS�ase wasdetermined (Fig. 3). The obtained data were con�firmed by rapid amplification of cDNA ends (RACE)using the Mint RACE primer set (Evrogen, Russia)according to manufacturer’s recommendations.

It was found that the full�length sAMC�ase cDNAsequence comprised 1362 bp. Rat sAMC�ase contains365 amino acid residues. The determined sequencewas deposited into the GenBank international data�base (accession KC529649).

The comparison of the structures of the long andshort forms of AMC�ase showed that the latter(sAMC�ase) is formed as a result of alternative splicingof the ChiA gene (Fig. 4).

Thus, this is the first study to determine the full�length cDNA sequence of sAMC�ase, which isexpressed in the rat respiratory lining in experimentalendotoxemia.

Specific primers used in the study

No. Primer no. Nucleotide sequence

1 AMC_forv 5'�AAGCTCATTCTTGTCACAGGTC�3'

2 AMC_rev 5'�TTAGTTTCATGGCCAGTTGC�3'

3 3' UTR�AMC_forv 5'�TTCTGGCACTGCATCAATGG�3'

4 5' UTR�AMC_rev 5'�CAGGAGAGTTTTCAGTTTGC�3'

531

42

Fig. 2. Nucleotide sequence of the sAMC�ase cDNA amplification product on a circular template comprising the 5'� and 3'�untranslated regions. Designations: 1—polylinker sequence of the pGEM�T vector; 2—sequences of the primers 3'UTR�AMC_forv and 5'UTR�AMC_rev; 3—the region of the known sequence encoding the C�terminal fragment of the mature ratAMC�ase (large font indicates the stop codon TGA); 4—sequences of primers CDS�1 adapter and PlugOligo�1 adapter (Evro�gen, Russian), which were used for the synthesis of total ds�cDNA; 5—5'� and 3'�untranslated sequences of the full�lengthsAMC�ase cDNA. The boundaries of the 375�bp fragment cloned into the pGEM�T vector (\ … \) and the site of ligation of ds�cDNA ends in the reaction of inverted ligation (../..) are shown.

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CLONING AND SEQUENCE ANALYSIS OF cDNA ENCODING 243

Fig. 3.Full�length cDNA sequence and the deduced amino acid sequence of the sAMC�ase protein. The 5'�untranslated region(highlighted in gray), the start codon ATG (shown in bold italic), the coding region, the stop codon TGA (shown is bold italic),the 3'�untranslated region (highlighted in gray), the polyadenylation site (underlined by the dotted line), and the Kozak sequence(in frame) are shown.

Fig. 4. Schematic representation of (I) long and (II) short forms of AMC�ase cDNA. The nucleotide sequence of the short formdiffers from that of the long form by the absence of the fragment comprising base pairs 73–274. Gray background shows the 5'�and 3'�untranslated regions. The consensus regions that ensure the splicing of the mRNA of the short form of rat AMC�ase areunderlined.

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ACKNOWLEDGMENTS

This work was supported by the Program of thePresidium of the Russian Academy of Sciences“Molecular and Cellular Biology” (V.M. Lipkin), andthe Russian Foundation for Basic Research (projectnos. 11�04�00761 and 12�04�31102).

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Translated by M. Batrukova