Cloning and characterization of a novel human TEKTIN1 gene

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Abstract

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.

Introduction

The microtubule cytoskeleton is essential for many cellular functions, including the formation of centrosomes, centrioles, mitotic spindles, basal bodies, cilia, and flagella. The main structural components of the microtubules are the α-and β-tubulin proteins. ɣ-and δ-tubulins have also been identified, which are generally restricted in their associations to centrosomes and basal bodies respectively. The structure and function of different microtubule systems depend on different isoforms of α-and β-tubulin, post-translational modifications of tubulin and a large number of microtubule-associated proteins [1]. One group of these proteins is the tektins, a family of at least three members (A, ∼53 kDa; B, ∼51 kDa; C, ∼47 kDa) from sea-urchin sperm [2]. Tektins are coiled-coil proteins that form filaments in the walls of ciliary and flagellar microtubules [3], [4].

Tektins form specialized filaments associated with the nine outer doublet microtubules of cilia and flagella. More specifically, tektin filaments are associated with a set of three chemically stable protofilaments of the A-microtubule wall, located near the inner junction with the B-tubule [5], [6]. Tektin filaments are composed of core protofilaments, which are assembled from tektin A and B heterodimers, and tektin C is suggested to form homodimers assembled onto the periphery of these core protofilaments or to form a second separate tektin filament [7]. The association of tektins and tubulin may provide the basis of the high stability of doublet microtubules. In addition, the location of tektin filaments in A-tubules and their periodic longitudinal spacings suggest that they may provide positional information for the attachment of inner dynein arms, radial spokes, nexin links. As predicted from their structural roles, all three sea-urchin tektins, A, B and C, are coordinately expressed during ciliogenesis. In addition to the role of tektins in ciliary structure, immunoblot and immunofluorescence studies with species ranging from sea urchins and mollusks to humans strongly imply that tektin filaments extend from similar or identical filaments in basal bodies and centrioles [8]. The cDNA sequences of sea urchin tektins A, B, C and mouse, rat, dog tektins [1], [4], [8], [9], [10], [11], [12], [13] provide further structural information for modeling interactions among the tektins and their subsequent associations within the tubulin lattice.

Recently, studies demonstrate that mouse tektin is involved in flagellar formation during male germ-cell development [1]. The development of the sperm flagella is a major event in spermiogenesis. Mammalian spermatogenesis takes place in the seminiferous tubules of the testis, which consists of a series of convoluted loops that have their ends connected in the excurrent duct system [14]. During spermiogenesis, the acrosomal structure forms at the apical end of the sperm head, whereas the centrioles become localized to the caudal end [15]. The centrioles then act as basal bodies for the formation of the axoneme of sperm tail [16], [17].

Although, the sperm flagellum varies in each species, the primary structural protein has been suggested to be similar. Tektins are components of flagellar microtubules, and most of the characterization has been done on sperm flagella of sea urchin [4]. Recently, mouse tektin studies indicate that tektins take part in the assembly of the basal body, which is the template for the assmebly of the falgellar axonemal microtubules of the maturing spermatozoa [1]. During the large-scale sequencing analysis of the human fetal brain cDNA library, we isolated a novel human tektin gene. Its amino acid sequence shows 82% identity to the mouse tektin1 protein; we term this gene TEKTIN1, in agreement with the Human Genome Organization (HUGO) Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature). Here, we report the cloning and characterization of the human TEKTIN1 gene, together with data on its genomic structure, tissue distribution and others.

Section snippets

cDNA library construction and DNA sequencing

A cDNA library was constructed in a modified pBluescript II SK (+) vector with the human fetal brain mRNA purchased from Clontech. The modified vector was constructed by introducing 2 SfiI recognition sites, i.e. SfiIA (5′-GGCCATTATGGCC-3′) and SfiIB (5′-GGCCGCCTCGGCC-3′), between the EcoRI and NotI sites of pBluescript II SK (+) (Stratagene). Double-stranded cDNAs were synthesized using SMART™ cDNA Library Construction Kit (Clontech) following the manufacturer's instructions. The cDNA inserts

Cloning and sequence analysis of human TEKTIN1 gene

From the human fetal cDNA library that we constructed, a novel cDNA was cloned. The nucleotide sequence and deduced amino acid sequence of this gene are shown in Fig. 1. The entire human cDNA is 1375 bp, containing an ORF from 60 to 1316, encoding 418 amino acid residues. The molecular mass and isoelectric point of the predicted translation product were calculated to be 48.3 kDa and 5.98, respectively. The polyadenylation signal, ATTAAA, is located at 1340 bp and 16 bp upstream of the poly(A)

Discussion

From earlier studies on sea urchins, tektin proteins were shown to be highly coiled-coil proteins that form filaments in the walls of ciliary and flagellar microtubules; they may also present in centrioles, centrosomes and mitotic spindles. Their predicted polypeotide structures suggest that tektins act as molecular templates and rulers in the assembly of cilia, flagella and their parent structures, basal bodies and centrioles [3], [4]. The first mammalian tektin, Tekt1, was cloned from a mouse

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Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank™ data base with the accession number AF357879.

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