The expression pattern of the C-terminal kinesin gene kifc1 during the spermatogenesis of Sepiella maindroni

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 2432 bp 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 structures, 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, the 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.     

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5′-untranslated region (5′-UTR), and a 107 bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.     

Molecular cloning and characterization of an Hsp90/70 organizing protein gene from Frankliniella occidentalis (Insecta: Thysanoptera,Thripidae)

The heat shock 90/70 organizing protein (Hop), also known as Sti-1 (stress-induced protein-1), is a co-chaperone that usually mediates the interaction of Hsp90 and Hsp70 and has been extensively characterized in mammals and plants. However, its role in insects remains unknown. In the present study, we isolated and characterized a Hop homologue gene from Frankliniella occidentalis (Fohop). The Fohop contains a 1659 bp ORF encoding a protein of 552 amino acids with a caculated molecular mass of approximately 62.25 kDa, which displays a reasonable degree of identity with the known Hops and shares several canonical motifs, including three tetratricopeptide repeated motif domains (TPR1, TPR2A and TPR2B) and two aspartic acid–proline (DP) repeat motifs (DP1 and DP2). As in other hops, Fohop contains introns, but the number and the position are quite variable. The mRNA expression patterns indicated that Fohop was constitutively expressed throughout the developmental stages, but was obviously upregulated by heat stress both in larvae and adults. Our studies imply that Hop, as in other Hsps, may play an important role in heat shock response of F. occidentalis.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

A thermostable cysteine protease precursor from a tropical plant contains an unusual C-terminal propeptide: cDNA cloning, sequence comparison and molecular modeling studies

We report here the cloning and characterization of the entire cDNA of a papain-like cysteine protease from a tropical flowering plant. The 1098-bp ORF of the cDNA codify a protease precursor having a signal peptide of 19 amino acids, a cathepsin-L like N-terminal proregion of 114 amino acids, a mature enzyme part of 208 amino acids and a C-terminal proregion of 24 amino acids. The derived amino acid sequence of the mature part tallies with the thermostable cysteine protease Ervatamin-C--as was aimed at. The C-terminal proregion of the protease has altogether a different sequence pattern not observed in other members of the family and it contains a negatively charged helical zone. The three-dimensional model of the precursor, based on the homology modeling and X-ray structure, shows that the extended peptide stretch region of the N-terminal propeptide, covering the interdomain cleft, contains protruding side chains of positively charged residues. This study also indicates that the negatively charged zone of C-terminal propeptide may interact with the positively charged zone of the N-terminal propeptide in a cooperative manner in the maturation process of this enzyme.     

The Drosophila homologue of the dystrophin gene - introns containing promoters are the major contributors to the large size of the gene

We show that the drosophila gene encoding the dystrophin-like protein (DLP) is as complex as the mammalian dystrophin gene. Three 5' promoters and three internal promoters regulate the expression of three full-length and three truncated products, respectively. The existence of this complex gene structure in such evolutionary remote organisms suggests that both types of products have diverse important functions. The promoters of both the DLP gene and the mammalian dystrophin gene are located in very large introns. These introns contribute significantly to the large size of the genes. The possible relevance of the conservation of the large size of introns containing promoters to the regulation of promoter activity is discussed.     

Functional diversification of kir7.1 in cichlids accelerated by gene duplication

Mutation in the inward rectifier potassium channel gene, kir7.1, was previously identified as being responsible for the broader stripe zebrafish skin pattern mutant, jaguar/obelix. An amino acid substitution in this channel causes a broader stripe pattern than that of wild type zebrafish. In this study we analyzed cichlid homologs of the zebrafish kir7.1 gene. We identified two kinds of homologous genes in cichlids and named them cikir7.1 and cikir7.2. Southern hybridization using cichlid genome revealed that cichlids from the African Great Lakes, South America and Madagascar have two copies of the gene. Cichlids from Sri Lanka, however, showed only one band in this experiment. Database analysis revealed that only one copy of the kir7.1 gene exists in the genomes of the teleosts zebrafish, tetraodon, takifugu, medaka and stickleback. The deduced amino acid sequence of cikir7.1 is highly conserved among African cichlids, whereas that of cikir7.2 has several amino acid substitutions even in conserved transmembrane domains. Gene expression analysis revealed that cikir7.1 is expressed specifically in brain and eye, and cikir7.2 in testis and ovary; zebrafish kir7.1, however, is expressed in brain, eye, skin, caudal fin, testis and ovary. These results suggest that gene duplication of the cichlid kir7.1 occurred in a common ancestor of the family Cichlidae, that the function of parental kir7.1 was then divided into two genes, cikir7.1 and cikir7.2, and that the evolutionary rate of cikir7.2 might have been accelerated, thereby effecting functional diversification in the cichlid lineage. Thus, the evolution of kir7.1 genes in cichlids provides a typical example of gene duplication--one gene is conserved while the other becomes specialized for a novel function.     

Identification and mRNA expression profile of glutamate receptor-like gene in quinclorac-resistant and susceptible Echinochloa crus-galli

Animal ionotropic glutamate receptors (iGluRs) function as Ca^2 + ion channels during excitatory neurotransmission in nerve cells. Here, a glutamate receptor-like gene (GLR) was identified and characterized from a plant — Echinochloa crus-galli. The GLR gene was designated EcGLR1 with GenBank no: JX518597. It has a 2793 bp open reading frame predicted to encode a 101.7 kDa protein. Sequence alignment showed that EcGLR1 is a GLR homologue. Its expression in response to quinclorac treatment was assessed by real-time PCR in near-isogenic lines of quinclorac-resistant (R) and susceptible (S) biotypes of E. crus-galli. The expression of EcGLR1 in the seedling leaf and root at least increased 5 times in the S plants and 22 times in the R plants after exposure to quinclorac. In the adult plant leaves, roots and stems, its expression increased 11–14 times in the S plants and 23–25 times in the R plants after quinclorac stimulation. In the seed, its expression was 4 times less in the S plants than that in the R plants, but after treatment, the levels all increased by about 24 times in the two biotypes. EcGLR1 expression was 1–4 times greater in the R plants than in that in the S plants, and after treatment by quinclorac, the difference increased to a ratio of 4 to 9. Its expression was higher in all tissues tested of R biotypes than in that of S plants before or after quinclorac treatment. The results of this study provide basic information for the further research of function of the EcGLR1 in resistance to quinclorac in E. crus-galli.     

Ankyrin-G in skeletal muscle: tissue-specific alternative splicing contributes to the complexity of the sarcolemmal cytoskeleton

Ankyrins are versatile adaptor proteins that join the spectrin-based cytoskeleton to transmembrane proteins, and have roles in organizing the microstructure of cell membranes. Molecular diversity of ankyrins in mammals arises from extensive alternative splicing of the products of three genes. There has been no systematic analysis of the diversity of expression of ankyrins-G, the widely expressed Ank3 gene products, in a complex tissue. We previously described Ank(G107), the first muscle-specific ankyrin-G. Here, we combined cDNA and database analyses to gain novel insight into the ankyrins-G of skeletal muscle. We find: (i) that Ank3 is composed of at least 53 exons, many of which are subject to tissue-specific splicing; (ii) five novel full-length cDNAs encoding two canonical (Ank(G197), Ank(G217)) and three small isoforms (Ank(G109), Ank(G128), Ank(G130)) bring to six the number of ankyrins-G expressed in skeletal muscle; (iii) a 76-residue insert in the C-terminal domain is a 'signature' for muscle ankyrins; (iv) variably spliced sequences of 17/18 and 195 residues increase diversity in the C-terminal domains. Comparison of endogenous ankyrins-G with in vitro translated cDNAs revealed that small ankyrins account for the majority of the immunoreactivity for ankyrin-G in soleus muscle. The small ankyrins, when expressed in vivo in the rat muscle, are all targeted to sarcolemmal costameres. Our results demonstrate the tissue-dependent alternative splicing of Ank3 in skeletal muscle and point to novel functions of small ankyrins-G in organizing microdomains of the plasma membrane.