Exon-1 skipping and intron-1 retaining by alternative splicing of the c-KIT gene encodes a novel splice variant in the skin of Merino sheep (Ovis aries).

c-KIT, a type III receptor protein tyrosine kinase, plays an essential role in melanocyte development, migration, and survival. Mutations within the c-KIT gene are previously shown to cause the white coat color phenotypes in pigs, mice, goats, and humans. However, up so far, the splicing isoform(s), genomic architecture of c-KIT have not been characterized well in merino sheep. Reverse transcriptase (RT)-PCR analysis with molecular prediction identified two basic splice variants: Transcript Variant-1, 2 for 12 bp insertion coding sequences (CDS) corresponding to the four amino acids 'GNSK', respectively. Using 5' RACE, here we report for the first time a novel c-KIT 'Transcript Variant-3' from the skin of merino sheep by comparative genome analyses at exon(1)-intron(1)-exon(2) boundaries. In contrast, a single product of 795 bp was characterized by 3' RACE. We also demonstrated that the c-KIT gene expression at the transcript level is not mediated via an intron-9 splicing event. Overall, beyond what was observed in other mammals, our data provide novel insights into the molecular structure of the c-KIT gene in sheep.

Description and Molecular Phylogeny of a Novel Hypotrich Ciliate from the Soil of Marche Region, Italy; Including Notes on the MOSYSS Project.

The morphology and morphogenesis during cell division of a new stylonychine hypotrich, Rigidocortex quadrinucleatus n. sp., were investigated using live observation and protargol staining. The new species was isolated from soil samples collected from an organic farm in the Marche Region, Italy, in framework of the MOSYSS project. Rigidocortex quadrinucleatus is characterized as follows: cell size about 180 × 80 µm in vivo; four ellipsoidal macronuclear nodules; 44 adoral membranelles: 18 fronto-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse, and five transverse cirri; dorsal kinety 3 with multiple fragmentation; resting cyst with hyaline ridges. Rigidocortex quadrinucleatus mainly differs from the type species R. octonucleatus in having four (vs. eight) macronuclear nodules. Rigidocortex quadrinucleatus can be easily confused with Sterkiella cavicola since both have a rather similar ventral ciliature; however, they can be separated by the slightly higher number of cirri in the left marginal row that runs along the posterior cell's margin in R. quadrinucleatus. Morphogenesis on the ventral surface is highly similar to that of Sterkiella species, but differs significantly on the dorsal surface (multiple vs. simple fragmentation of dorsal kinety 3). Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to S. cavicola.

Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Two Gonostomatid Ciliates from the Soil of Lombardia, Italy; including Note on the Soil Mapping Project.

Two gonostomatid ciliates, Gonostomum paronense n. sp. and G. strenuum, isolated from the soil sample of paddy field, Lombardia, Italy, were investigated using live observation and protargol impregnation. Gonostomum paronense n. sp. is mainly characterized by a tailed body, frontoventral cirri arranged in pairs, and presence of pretransverse and transverse cirri. Morphologically and morphometrically, the new species is similar to Gonostomum namibiense in having a tailed body and frontoventral cirral pairs; however, it differs mainly in the number of frontoventral cirral pairs (seven vs. three). Phylogenetic analyses based on the SSU rDNA sequences show that the new species is more closely related to G. namibiense than to G. strenuum, supporting the morphological classification based on the cirral pattern and the tailed body. However, due to the poor nodal support and absence of gene sequence of the type species Gonostomum, a more robust phylogeny of this group still remains unresolved. The biometric data of the Italian population of Gonostomum strenuum overlap with those from other known populations. Both species were collected from the industrial area of Parona, in the framework of the "Soil Mapping, Lombardia" project in which, for the first time in Italy, soil ciliates were used as bioindicators of soil quality.

UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii.

The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

The alpaca melanocortin 1 receptor: gene mutations, transcripts, and relative levels of expression in ventral skin biopsies.

The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5'-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T)) were observed in coding region and three in the 3'UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation.

Morphology, morphogenesis and molecular phylogeny of a novel soil ciliate, Pseudouroleptus plestiensis n. sp. (Ciliophora, Oxytrichidae), from the uplands of Colfiorito, Italy.

The terrestrial oxytrichid ciliate Pseudouroleptus plestiensis n. sp., isolated from soil samples collected from the uplands of Colfiorito (Umbria region, Italy), was investigated using live observation and protargol impregnation. The morphology, morphogenesis and molecular phylogeny inferred from small-subunit (SSU) rRNA gene sequences were studied. The novel species is mainly characterized by the following: a cell size of about 145×35 µm in vivo; two ellipsoidal macronuclear nodules and two to four micronuclei; adoral zone about 26% of body length with a mean of 30 membranelles; about 40 cirri in the right marginal row and 38 in the left marginal row; left fronto-ventral row consisting of about 27-40 cirri, right fronto-ventral row of about three to seven cirri forming a short row to the right of the rear portion of the left fronto-ventral row; one parabuccal cirrus ( = III/2), one buccal and one post-peristomial cirrus; and four dorsal kineties with caudal cirri at the end of kineties 1 and 2. The morphogenesis of the novel species is similar to that of Pseudouroleptus caudatus. Phylogenetic analyses based on SSU rRNA gene sequences consistently placed the novel species within the family Oxytrichidae Ehrenberg, 1838, clustering with P. caudatus and the genus Strongylidium. The results from the present study contribute to the expanding knowledge of the diversity of ciliates in Italian soil.

Morphology, morphogenesis, and molecular phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a "Stylonychine Oxytrichidae" (Ciliophora, Hypotrichida) without transverse cirri.

A terrestrial oxytrichid ciliate Paraparentocirrus sibillinensis n. gen., n. sp., which was found in soil samples of a beech forest stand within the National Park of Sibillini Mountains, Italy, was investigated using live observation and protargol impregnation. The morphology of interphase, morphogenesis, and molecular phylogeny inferred from SSU rDNA sequences of this ciliate were studied. Paraparentocirrus n. gen., is mainly characterized by a semirigid body, an undulating membrane in the Oxytricha pattern, six fronto-ventral (FV) rows, the absence of transverse cirri, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kinety 4. During morphogenesis, oral primordia develop through the proliferation of basal bodies from some cirri of FV rows 4 and 5, and FV row 6 takes part in the anlagen formation of the proter. The dorsal morphogenesis was typical of oxytrichids, with simple fragmentation of dorsal kinety 3, and the dorsomarginal rows developed from the right marginal row. Phylogenetic analyses based on the SSU rDNA sequences support the classification of this new genus in the stylonychines.

Chemical offense by means of toxicysts in the freshwater ciliate, Coleps hirtus.

Coleps hirtus is a small common freshwater ciliate belonging to the protostomatid group, its body covered by calcified plates assembled to form an armor. Coleps feeds on bacteria, algae, flagellates, living and dead ciliates, animal and plant tissues. To assist its carnivorous feeding the ciliate is equipped with offensive extrusomes (toxicysts), clustering mainly in and around its oral aperture. In this study, we isolated the discharge of the toxicysts from living cells, evaluating its cytotoxic effects against various ciliate species, and demonstrating that it is essential for the effectiveness of Coleps' predatory behavior. The analysis of the toxicyst discharge performed by liquid chromatography-electrospray-mass spectrometry and gas chromatography-mass spectrometry, revealed the presence of a mixture of 19 saturated, monounsaturated and polyunsaturated free fatty acids with the addition of a minor amount of a diterpenoid (phytanic acid).

Combination of two regulatory elements in the Tetrahymena thermophila HSP70-1 gene controls heat shock activation.

The induction of heat shock genes (HSPs) is thought to be primarily regulated by heat shock transcription factors (HSFs), which bind target sequences on HSP promoters, called heat shock elements (HSEs). In this study, we investigated the 5' untranslated regions of the Tetrahymena thermophila HSP70-1 gene, and we found, in addition to the canonical and divergent HSEs, multiple sets of GATA elements that have not been reported previously in protozoa. By means of in vivo analysis of a green fluorescent protein reporter transgene driven by the HSP70-1 promoter, we demonstrate that HSEs do not represent the minimal regulatory elements for heat shock induction, since the HSP70-1 is tightly regulated by both HSE and GATA elements. Electrophoretic mobility shift assay also showed that HSFs are constitutively bound to the HSEs, whereas GATA elements are engaged only after heat shock. This is the first demonstration by in vivo analysis of functional HSE and GATA elements in protozoa. Furthermore, we provide evidence of a functional link between HSE and GATA elements in the activation of the heat shock response.

Morphology and ontogeny of Tetmemena pustulata indica nov. subspec. (Ciliophora, Hypotricha), from the Thane Creek, Mumbai, India.

The morphology and ontogenesis of a novel stylonychid ciliate, Tetmemena pustulata indica nov. subspec., were investigated using live observation and protargol preparation. The new subspecies was isolated from a water sample collected from the Thane Creek, Mumbai, India. At first glance, T. pustulata indica looked very similar in morphology and ontogenesis to the well-known Tetmemena pustulata, however, on detailed investigation some non-overlapping features in the ciliature were identified, i.e., the numbers of cirri in marginal rows, adoral membranelles, and bristles in dorsal kineties. These morphometric differences justified the separation at subspecies level. Furthermore, the resting cysts are different, i.e., with smooth external layer in T. pustulata indica whereas spinous in the nominotypical subspecies T. pustulata pustulata. The Venezuelan population of T. pustulata described by Foissner corresponds very well with the Indian population in the ciliature; however, the information on the resting cyst is lacking for the former. Our study presents an example of a cyst subspecies among hypotrich ciliates, and thus extends the concept which has been mainly reported for spathidiids.

PDGFA in Cashmere Goat: A Motivation for the Hair Follicle Stem Cells to Activate.

The cashmere hair follicle (HF) perpetually goes through cycles of growth, involution and rest. The photoperiod is the main factor in the control of seasonal coat change in cashmere goats while stem cells play a crucial role in the HF growth. Several factors, including Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) are implicated in HF morphogenesis and cycle. In this work, the mentioned molecules were investigated to evaluate their role in follicular cycle activation. The study was performed on skin samples collected at different periods of HF cycle and the molecular expression of PDGFA, BMP2 and LHX2 was evaluated by Real-Time PCR (qPCR) at each time point. Since PDGFA showed the most variation, the goat PDGFA gene was sequenced and the protein localization was investigated by immunohistochemistry together with PDGF receptor α (PDGFRα). PDGFA immunostaining was observed in the basal layer of the HF outer root sheath and the immunoreaction appeared stronger in the regressive HFs compared to those in the anagen phase according to qPCR analysis. PDGFRα was observed in the HF epithelium, proving the effect of PDGFA on the follicular structure. The data obtained suggest that PDGFA and BMP2 are both implicated in HF cycle in goat. In particular, PDGFA secreted by the HF is involved in the anagen activation.

Shedding light on the polyphyletic behavior of the genus Sterkiella: The importance of ontogenetic and molecular phylogenetic approaches.

Present study, investigates a poorly known species of the genus Sterkiella, i.e., S. tricirrata, based on two populations isolated from soil samples collected from the Colfiorito Regional Park, Umbria Region, Italy and from the Silent Valley National Park, India. Both populations showed a highly similar morphology, however different ontogenetic pattern in between. The study confirms the validity of the species S. tricirrata which was considered to be a species within the Sterkiella histriomuscorum complex. The main ontogenetic difference between S. tricirrata and other species of the genus Sterkiella is the different mode of formation of anlagen V and VI of the proter in the former. In the phylogenetic analyses, Sterkiella tricirrata clusters with Sterkiella sinica within the stylonychine oxytrichids, in a clade away from the type species (Sterkiella cavicola) of the genus Sterkiella. The study highlights the importance of ontogenetic as well as molecular data in shedding light on the polyphyletic behavior of the genus Sterkiella. A detailed description of S. tricirrata based on morphology, ontogenesis and molecular phylogenetic methods is presented. Further, the improved diagnosis has been provided for the genus Sterkiella and the poorly known species S. tricirrata.

Evidence of post-transcriptional readthrough regulation in FGF5 gene of alpaca.

Two different phenotypes are described in alpaca, identified as suri and huacaya, which differ in the type of fleece. The huacaya fleece is characterized by compact, soft and highly crimped fibers, while the suri fleece is longer, straight, less-crimped and lustrous. In our study, the Fibroblast growth factor 5 (FGF5) was investigated as a possible candidate gene for hair length in alpaca (Vicugna pacos). As previously identified in other mammals, our results show that the alpaca FGF5 gene gives rise to a short (FGF5S) and a long (FGF5) isoform. Interestingly, in the long isoform, we observed a point mutation (i.e., a transition C>T at position 499 downstream of the ATG codon) that is able to generate a premature termination codon (PTC). The highly conserved nucleotide and amino acid sequence after PTC suggested a readthrough event (RT) that was confirmed by western blot analysis. The analysis of cDNA sequence revealed motifs and structures of mRNA undergoing RT. In fact, the event is positively influenced by particular signals harbored by the transcript. To the best of our knowledge, this is the first case of a readthrough event on PTC reported for the FGF5 gene and the first case of this translational mechanism in alpaca.

Rigidosticha italiensis n. gen., n. sp. (Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy.

The morphology of Rigidosticha italiensis n. gen., n. sp., which was found in a soil sample collected from an uncultivated field in Lombardia, Italy, was investigated using live observation and protargol staining. Rigidosticha n. gen. is characterised by a rigid body, undulating membranes resembling a Steinia pattern, oxytrichid frontal ciliature, distinct mid-ventral cirral pairs, transverse cirri, one right and one left row of marginal cirri, absence of dorsal kinety 3 fragmentation, more than two dorsomarginal rows, and caudal cirri. The new species shows the following features: size in vivo 230-330×100-170µm, on average 230×115µm in protargol preparations; two ellipsoidal macronuclear nodules; 51 adoral membranelles; one buccal cirrus; one parabuccal cirrus; two frontoterminal cirri; 16 mid-ventral cirral pairs; and three transverse cirri. Rigidosticha mainly differs from Rigidothrix, Afrophrya, Uroleptus, and Territricha, in having the undulating membranes in Rigidosticha (vs. oxytrichid and cyrtohymenid) pattern. The oxytrichid frontal ciliature and midventral pattern in the present species further support the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis.

Fine-tune investigations on three stylonychid (Ciliophora, Hypotricha) ciliates.

The morphology and morphogenesis of two stylonychid ciliates, Stylonychia ammermanniGupta et al., 2001 and Tetmemena bifaria (Stokes, 1887) Berger, 2001, isolated from soil samples of Lombardia region, Northern Italy, and water samples from Cheonggancheon stream, South Korea, were investigated. The Italian population of S. ammermanni was found to be very similar in morphology to the Indian type population and the junior synonym S. harbinensisShi and Ammermann, 2004 since most of the morphometric data overlapped. On the contrary, the Korean population of S. ammermanni showed some non-overlapping differences in ciliature suggesting a separation at subspecies level, i.e., S. ammermanni ammermanni and S. ammermanni koreana nov. subspec. Furthermore, the resting cyst of the Italian population of S. ammermanni has many ring-shaped structures on the surface which, however, were not observed in the Korean population. Phylogenetic analyses based on the SSU rRNA gene sequences show that the Italian and Korean populations of S. ammermanni fit well into the S. mytilus complex and moderately support the subspecies separation. Additionally, we split Tetmemena bifaria into two subspecies, viz., T. bifaria bifaria and T. bifaria minima nov. subspec. based on differences in the number of adoral membranelles and number of cirri in the marginal rows of the Italian and the Argentinian populations in comparison with the populations described by Wirnsberger et al. (1985).

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India.

The morphology and morphogenesis during cell division of Sterkiella tetracirrata n. sp., isolated from a soil sample collected from the Silent Valley National Park, Kerala, India, were investigated using live observation, protargol staining and scanning electron microscopy. The new species differs from its congeners by the following combination of features: cell size in vivo 85-110×35-50µm, on average 84×37µm in protargol preparations; four ellipsoidal macronuclear nodules; 31 adoral membranelles; 17 frontal-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse and invariably four transverse cirri; resting cyst with separate macronuclear nodules. Sterkiella tetracirrata differs from the similar species S. terricola in the number of transverse cirri (invariably 4 vs. 3) and in the number of adoral membranelles (24-35 vs. 22 or 23). Morphogenesis resembles that of its congeners S. nova and S. histriomuscorum. Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to either S. cavicola or S. histriomuscorum. The analyses support the morphological evidence (e.g., similarity in the oral apparatus and the dorsal kinety pattern) that Gastrostyla and Pattersoniella evolved from a Sterkiella-like ancestor.

Biochemical characterization and quantitative gene expression analysis of the multi-stress inducible metallothionein from Tetrahymena thermophila.

A cadmium-binding protein with biochemical features of a metallothionein (MT) has been isolated and purified to homogeneity from the ciliate Tetrahymena thermophila. N-terminal sequencing revealed the posttranslational cleavage of the first two amino acids and, in general, a high degree of identity with known MTs from other ciliates. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis of the apothionein revealed a molecular mass (16,763 Da) higher to those of mammals and of other protozoa. Finally, quantitative real-time PCR has been used to investigate the susceptibility of this ciliate MT to gene activation in response to heavy metals and to other stressors. Our data indicate that while zinc is not effective at all and cadmium is the best inducer, other stress factors, such as mercury, copper, heat and hydrogen peroxide, also activated gene transcription. As in vertebrate cells, interleukin-6 (IL-6) that stimulates ciliate growth, was able to enhance MT gene synthesis. This complex of data seems to indicate a general role of this protein in stress response.

Alternative splicing of the sheep MITF gene: novel transcripts detectable in skin.

Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor, which regulates the differentiation and development of melanocytes and pigment cell-specific transcription of the melanogenesis enzyme genes. Though multiple splice variants of MITF have been reported in humans, mice and other vertebrate species, in merino sheep (Ovis aries), MITF gene splicing has not yet been investigated until now. To investigate the sheep MITF isoforms, the full length mRNA/cDNAs from the skin of merino sheep were cloned, sequenced and characterized. Reverse transcriptase (RT)-PCR analysis and molecular prediction revealed two basic splice variants with (+) and without (-) an 18 bp insertion viz. CGTGTATTTTCCCCACAG, in the coding region (CDS) for the amino acids 'ACIFPT'. It was further confirmed by the complete nucleotide sequencing of splice junction covering intron-6 (2463 bp), wherein an 18bp intronic sequence is retained into the CDS of MITF (+) isoform. Further, full-length cDNA libraries were enriched by the method of 5' and 3' rapid amplification of cDNA ends (RACE-PCR). A total of seven sheep MITF splice variants, with distinct N-terminus sequences such as MITF-A, B, E, H, and M, the counterparts of human and mouse MITF, were identified by 5' RACE. The other two 5' RACE products were found to be novel splice variants of MITF and represented as 'MITF truncated form (Trn)-1, 2'. These alternative splice (AS) variants were illustrated using comparative genome analysis. By means of 3' RACE three different MITF 3' UTRs (625, 1083, 3167bp) were identified and characterized. We also demonstrated that the MITF gene expression determined at transcript level is mediated via an intron-6 splicing event. Here we summarize for the first time, the expression of seven MITF splice variants with three distinct 3' UTRs in the skin of merino sheep. Our data refine the structure of the MITF gene in sheep beyond what was previously known in humans, mice, dogs and other mammals.