Molecular cloning and characterization of KIFC1-like kinesin gene (ot-kifc1) from Octopus tankahkeei.

Faculty of Life Science and Bioengineering, Ningbo University, Zhejiang 315211, PR China.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology (Impact Factor: 1.61). 03/2010; 156(3):174-82. DOI: 10.1016/j.cbpb.2010.03.004
Source: PubMed

ABSTRACT Spermiogenesis in Octopus tankahkeei involves striking cellular reorganization to generate a mature spermatozoon. This process may require spermatid-specific adaptation of cytoskeleton and associated molecular motor proteins. KIFC1 is a C-terminal kinesin motor with important roles in acrosome biogenesis and nuclear reshaping during spermiogenesis in rat. Here, we have cloned and characterized the gene encoding a homologue of rat KIFC1, termed as ot-kifc1, from the testis of O. tankahkeei. The 2229 bp complete cDNA contains a 75 bp 5'-untranslated region, a 1992 bp open reading frame and a 162 bp 3'-untranslated region. The deduced protein shares an overall identity of 40%, 41%, 39% and 41% with its counterpart from human, rat, mouse and African clawed frog, respectively. Tissue expression analysis revealed ot-kifc1 was expressed in testis, gill and hepatopancreas, but not in other tissues examined. In situ hybridization result showed the ot-kifc1 message was hardly detectable in early spermatid, concentrated at the tail region of intermediate spermatid, abundant in spermatid undergoing dramatic elongation and compression, enriched at one end in late spermatids and disappeared in mature sperm. In conclusion, the expression of ot-kifc1 at specific stages of spermiogenesis suggests a role for this motor in major cytological transformations.

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    ABSTRACT: The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339 bp that contained a 216 bp 5'-untranslated region (5'UTR), a 194 bp 3'-untranslated region (3'UTR) and a 1,929 bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71 kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116 aa, a stalk domain from 117 to 291 aa and a conserved carboxyl motor domain from 292 to 642 aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8 % identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.
    Molecular Biology Reports 09/2013; · 1.96 Impact Factor
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    ABSTRACT: Kinesin superfamily is a class of microtubule-dependent motors that play crucial roles in acrosome biogenesis, nuclear reshaping and flagellum formation during spermiogenesis. We have cloned kinesin-like gene kifc3 (termed ec-kifc3) from the total RNA of the testis of the skink Eumeces chinensis. The cDNA sequence of ec-kifc3 had a full-length of 3033bp, including a 260bp 5'-untranslated region (5'UTR), a 445bp 3'-untranslated region (3'UTR) and an open reading frame that encoded a 775-amino-acid protein. Additionally, the calculated molecular weight of the putative ec-KIFC3 was 87kDa and its estimated isoelectric point was 6.18. Structurally, the putative ec-KIFC3 had three domains: head domain, neck domain and tail domain. Protein alignment demonstrated that ec-KIFC3 had 47.2%, 67.8%, 68.8%, 69.3% and 76.8% identity with its homologues in Xenopus laevis, Mus musculus, Cricetulus griseus, Homo sapiens, and Gallus gallus. The phylogenetic analysis showed that ec-KIFC3 was more related to KIFC3 in vertebrates than invertebrates. Tissue expression results showed the presence of ec-KIFC3 in various tissues with its highest expression in the testis. In situ hybridization demonstrated that ec-KIFC3 mRNA was distributed around the nucleus in early and middle stage spermatids and expressed in the nucleus in the elongating spermatids during spermiogenesis. Besides, the ec-KIFC3 mRNA was expressed in the acrosome of the developmental spermatids. From the results of in situ hybridization and previous researches, we speculated that ec-KIFC3 may play a role in nuclear morphogenesis and acrosome formation during spermiogenesis of E. chinensis. Copyright © 2014. Published by Elsevier B.V.
    Gene 11/2014; · 2.20 Impact Factor
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    ABSTRACT: In this study, we investigated the gene sequence and characteristic of kifc1 in Sepiella maindroni through PCR and RACE technology. Our research aimed particularly at the spatio-temporal expression pattern of kifc1 in the developmental testis through in situ hybridization. The particular role of kifc1 in the spermatogenesis of S. maindroni was our particular interest. Based on multiple protein sequence alignments of KIFC1 homologues, kifc1 gene from the testis of S. maindroni was identified, which consisted of 2432bp including a 2109 in-frame ORF corresponding to 703 continuous amino acids. The encoded polypeptide shared highest similarity with Octopus tankahkeei. Through the prediction of the secondary and tertiary structure, the motor domain of KIFC1 was conserved at the C-terminal, having putative ATP-binding and microtubule-binding motifs, while the N-terminal was more specific to bind various cargoes for cellular events. The stalk domain connecting between the C-terminal and N-terminal, determined the direction of movement. According to RT-PCR results, the kifc1 gene is not tissue-specific, commonly detected in different tissues, for example, testis, liver, stomach, muscle, caecum and gills. Through an in situ hybridization method, the expression pattern of KIFC1 protein mimics in the spermatogenesis of S. maindroni. During the primary stage of the spermatogenesis, the kifc1 mRNA signal was barely detectable. At the early spermatids, the signal started to be present. With the elongation of spermatids, the signals increased substantially. It peaked and gathered around the acrosome area when the spermatids began to transform to spindle shape. As the spermatids developed into mature sperm, the signal vanished. In summary, the expression of kfic1 at specific stages during spermiogenesis and its distribution shed light on the potential functions of this motor in major cytological transformations. The KIFC1 homologue may provide a direct shaping force to the nucleus or influence the shaping process through indirect regulation.
    Gene 09/2013; · 2.20 Impact Factor


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May 22, 2014