Transcriptase-polymerase Chain Reaction Amplification Research Articles

Isolation and characterization of a FLOWERING LOCUS T homolog from pineapple (Ananas comosus (L.) Merr)

FLOWERING LOCUS T (FT)-like genes are crucial regulators of flowering in angiosperms. A homolog of FT, designated as AcFT (GenBank ID: HQ343233), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcFT is 915bp in length and contains an ORF of 534bp, which encodes a protein of 177 aa. Molecular weight was 19.9kDa and isoelectric point was 6.96. The deduced protein sequence of AcFT was 84% and 82% identical to homologs encoded by CgFT in Cymbidium goeringii and OgFT in Oncidium Gower Ramsey respectively. Quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AcFT was high in flesh and none in leaves. qRT-PCR analyses in different stages indicated that the expression of AcFT reached the highest level on 40 d after flower inducing, when the multiple fruit and floral organs were forming. The 35S::AcFT transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants.

Absence of Gastrointestinal Pathogens in Ileum Tissue Resected for Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal emergencies in premature infants and has been linked with viral antigens in as much as 40% of cases in single-center cohorts. We examined 28 tissue sections from surgically resected ileum from 27 preterm infants with NEC from 2 separate institutions for 15 common bacterial, viral, and parasitic gastrointestinal pathogens using multiplex reverse transcriptase polymerase chain reaction amplification and suspension array detection methods. We did not detect infectious enteritis pathogens in any of the NEC tissues and conclude that gastrointestinal pathogens are a rare cause of NEC.

Cloning and Expression Analysis of a PISTILLATA Homologous Gene from Pineapple (Ananas comosus L. Merr)

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants.

Multiple organ inflammatory response to portosystemic shunt in the rat

Portosystemic shunt surgery is the best procedure to prevent recurrent bleeding of esophageal varices, but carries a high risk of postoperative inflammatory complications, including hepatic encephalopathy. Thus, portosystemic shunting procedures could induce a systemic inflammatory response with multiple organ dysfunction syndrome, including hepatic encephalopathy. To verify this hypothesis we used male Wistar rats at 6weeks of postoperative evolution: Control (CR; n=14), Sham-operated (SO; n=8) and rats with end-to-side portacaval shunt (PCS; n=15). TNF-α, IL-1β and IL-10 were assayed by ELISA techniques, the expression of the endothelial constitutive nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), constitutive and inducible heme-oxygenase (HO-1 and HO-2) were assayed by Western-blot. mRNA levels of HO-1, HO-2, TNF-α, IL-1β and IL-10 were quantified by reverse transcriptase polymerase chain reaction amplification (RT-PCR) in the small bowel, liver, spleen and lungs. Portacaval shunting in the rat produces an interorgan imbalance of pro- and anti-inflammatory mediators. TNF-α mRNA expression is decreased in the liver (0.69±0.28, p<0.05). The hepatic production of IL-Iβ (204.13±71.90pg/100g; p<0.001) and IL-10 (4505.47±337.97pg/100g; p<0.001) is also decreased. However, the intestinal pro-inflammatory (TNF-α: 1471.86±153.62pg/100g, p<0.001; IL-1β: 48.35±9.84pg/100g, p<0.001 and iNOS: 0.59±0.01, p<0.01) and anti-inflammatory (IL-10: 1503.39±53.5pg/100g, p<0.001 and HO-1: 2.23±0.16, p<0.001) mediators are increased. Total portacaval shunting in the rat induces impairments of pro- and anti-inflammatory mediators in the splanchnic–lung axis that could be associated with a multiple organ dysfunction syndrome. Therefore, the complications after portosystemic shunts could be integrated into a systemic inflammatory response of possible intestinal origin.

Three β-galactosidase cDNA clones related to fruit ripening in papaya (Carica papaya)

β-Galactosidase (EC 3.2.1.23) is a hydrolase which plays an important role in cell wall modification and fruit softening during ripening. In this study, three full-length β-galactosidase cDNA clones were successfully obtained from papaya mesocarp using different approaches. pPGBII which is 2,771 bp in size, was isolated from a papaya ripe mesocarp cDNA library using a heterologous probe. The other two cDNA clones, pBG(a) and pBG(b), which are 3,168 and 2,580 bp in size, respectively, were amplified from ripe papaya fruit using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) approaches. The pPBGII, pBG(a) and pBG(b) cDNAs, respectively, were expected to yield a putative mature polypeptide of 79, 91 and 56 kDa with isoelectric points (pI) of 8.12, 8.75 and 8.26. The genomic DNA gel blot analysis indicated that all of the β-galactosidase genes exist as multiple copies in the papaya genome hence they belong to a multigene family. All three cDNA clones were expressed in fruits during ripening with varying patterns. In mesocarp, pPBGII mRNA was only expressed during ripening and peaked at the half-ripe stage when the fruits undergo dramatic softening. Meanwhile, pBG(a) mRNA expression increased from the immature green stage to the half-ripe stage where it reached maximum level before declining. pBG(b) mRNA level accumulated abundantly at the mature green stage and decreased thereafter. Therefore, we suggest that pPBGII and pBG(a) cDNA clones characterized in this work may be involved in fruit softening during papaya ripening while the fruit-specific pBG(b) may be related to early ripening stage.

Tobacco plants ectopically expressing the Ammopiptanthus mongolicus AmCBL1 gene display enhanced tolerance to multiple abiotic stresses

Ca2+ is a critical component in signal transduction pathways that lead to stress gene expression in higher plants and regulates a wide range of physiological processes. The calcineurin B-like protein (CBL) family represents a unique group of calcium sensors in plants, and plays a key role in decoding calcium transients. Here, a CBL1 homolog gene, AmCBL1 (NCBI accession no. AY902246), was isolated from Ammopiptanthus mongolicus (Maxim.) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Quantitative real-time PCR (QRT-PCR) experiments showed that AmCBL1 was significantly induced by drought, high salinity, heat, and CaCl2 treatments in A. mongolicus seedlings. Subcellular localization analysis suggested that AmCBL1 is a plasma membrane-localized protein. Our results suggest that AmCBL1 is not only a positive regulator of salt response but also a positive regulator of temperature stresses in A. mongolicus.

The ionotropic γ-aminobutyric acid receptor gene family of the silkworm, Bombyx mori

&gamma;-Aminobutyric acid (GABA) is a very important inhibitory neurotransmitter in both vertebrate and invertebrate nervous systems. GABA receptors (GABARs) are known to be the molecular targets of a class of insecticides. Members of the GABAR gene family of the silkworm, Bombyx mori, a model insect of Lepidoptera, have been identified and characterized in this study. All putative silkworm GABAR cDNAs were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Bombyx mori appears to have the largest insect GABAR gene family known to date, including three RDL, one LCCH3, and one GRD subunit. The silkworm RDL1 gene has RNA-editing sites, and the RDL1 and RDL3 genes possess alternative splicing. These mRNA modifications enhance the diversity of the silkworm's GABAR gene family. In addition, truncated transcripts were found for the RDL1 and LCCH3 genes. In particular, the three RDL subunits may have arisen from two duplication events.

Isolation and Characterization of the Betaine Aldehyde Dehydrogenase Gene in

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the glycine betaine from choline. The BADH gene from turfgrass Ophiopogon japonicus has not been reported. In this study, we first isolated the full length cDNA of betaine aldehyde dehydrogenase gene (OjBADH) from O. japonicus using Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) and Rapid Amplification of cDNA Ends (RACE) techniques. The OjBADH gene (GenBank accession number: DQ645888) has 1785 nucleotides with the 5’ untranscribed region (UTR) of 63 nucleotides, 3’ UTR of 219 nucleotides, and an open reading frame of 1503 nucleotides. This gene encodes a polypeptide of 500 amino acids. It shares a high homology with BADH genes of other Chenopodiaceae species. The putative protein includes a conservative region of phosphofructokinase, aldehyde dehydrogenase, and glutamy phosphoric acid reductase. Overexpression of OjBADH in transgenic tobacco plants demonstrated 2-2.5 folds increase of glycine betaine content and 60- 85% increase of survival rate under salt tolerance. These results suggested that the O. japonicus BADH gene may be used to engineer plants for salt stress tolerance.

Cloning of carnation GA 20-oxidase and the construction of a plant RNAi vector

AbstractA pair of degenerate primers was designed based on a conserved domain of GA 20-oxidase reported in other plants. The full-length (1179 bp) carnation (Dianthus caryophyllus L. cv. Master) GA 20-oxidase cDNA (named Dc20ox) was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). BLAST analysis revealed that the deduced amino acid sequence had high homology (66–75%) with the GA 20-oxidase sequences from other plants. An RNAi vector (pART400) was constructed from a 400 bp fragment representing a highly conserved region of GA 20-oxidase.

Increased ABCG2 Expression Could Be Responsible for Resistance to Imatinib Mesylate in Patients with Chronic Myeloid Leukemia Who Do Not Have Mutations in BCR-ABL Kinase Domain

Mutation in the Bcr-Abl kinase domain is one of the most common mechanism of resistance to imatinib mesylate (IM), seen in 30– 90% of patients with chronic myeloid leukemia (CML). A total of 57 patients with CML, 41 in chronic phase, 10 in accelerated phase and 6 in blast crisis, were analyzed for bcr-abl kinase domain (KD) mutations using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the bcr-abl KD followed by direct sequencing. Twelve different mutations including three novel mutations were identified in the bcr-abl KD in 18 patients. Resistance to IM was considered if the patient had rising white blood cell counts or did not achieve hematological remission at 6 months; or had rising bcr-abl levels (as determined by fluorescence in situ hybridization or real-time quantitative PCR) after the start of IM treatment. In order to evaluate other potential mechanisms of resistance in patients without mutations in the bcr-abl kinase domain, we tested the expression levels of transporter genes known to be involved in IM influx and efflux. mRNA expression levels of efflux transporters, ABCG2 and ABCB1, and influx transporter, hOCT1 were measured by real time quantitative PCR using ABL to normalize the expression levels of the same. Serially diluted cDNA made from HepG2 RNA was used to make the standard curve and amplification efficiencies of the target and the house keeping genes were similar. Each sample was analyzed in duplicate and the experiment was repeated twice. In the patients without mutations in the bcr-abl KD, significantly higher ABCG2 mRNA levels were observed compared to patients with mutations (Table). Transcript levels of ABCB1, hOCT1 or bcr-abl were not significantly different between the two groups. This study suggests that over expression of ABCG2 may be one of the mechanisms of resistance to imatinib in patients without mutations in bcr-abl. Future studies should not only compare the expression of these transporters at diagnosis (before the start of IM treatment) but also at the time of clinical resistance. This will help understand the influence of expression levels of these transporters in achieving haematological or molecular response to increased IM doses.Bcr-abl mutation positive (n=18)Bcr-abl mutation negative (n=39)p valueABCG2: median (range)0.0126 (0.0004–0.45)0.051 (0.0041–0.51)0.034ABCB1: median (range)0.1842 (0.00349–0.51)0.1819 (0.0463–0.68)NShOCT1: median (range)488 (3.4–3394)486 (62–3059)NSBCR-ABL: median (range)53.96 (5.73–179.1)51.3 (2.36–129)NS

A Putative Peroxidase cDNA from Turnip and Analysis of the Encoded Protein Sequence

A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

Cloning and Characterization of a Stearoyl–Acyl Carrier Protein Desaturase Gene from Cinnamomum longepaniculatum

The stearoyl–acyl carrier protein desaturase (SAD) is a key enzyme that determines the ratio of saturated to unsaturated fatty acids in higher plants. Using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends, a full-length cDNA of SAD was obtained from developing leaves of Cinnamomum longepaniculatum. Sequence analysis showed that the deduced amino acid sequence had high similarity to other reported SADs. The CSAD gene was functionally expressed in Escherichia coli, and the desaturating activity of the recombinant protein was markedly detected when assayed in vitro by adding spinach ferredoxin. Southern blot analysis indicated that the gene was a member of a small gene family. Northern blotting revealed that the CSAD gene was highly expressed in developing leaves of C. longepaniculatum. These results would provide opportunities for modifying fatty acid composition in C. longepaniculatum.

Impact of chicken thrombopoietin and its receptor c-Mpl on hematopoietic cell development

The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chicken TPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. Chicken TPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. Both chicken TPO and its receptor c-Mpl were identified and cloned. Expression of chicken TPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chicken TPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. The characterized chicken thrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.

PH but not hypoxia affects neonatal gene expression: Relevance for housekeeping gene selection

Objective. To identify a candidate neonatal housekeeping gene and to determine the effects of pH and PaO2 on the stability of newborn gene expression in physiologically hypoxic and acidotic newborn blood.Methods. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) amplification was performed for four commonly used housekeeping genes (GAPDH, β-actin, cyclophilin, 28S rRNA) on extracted RNA. Blood gas analyses determined pH and PaO2 levels.Results and conclusions. β-Actin was the least variable and GAPDH the most variable housekeeping gene studied. pH negatively correlated with gene expression levels. PaO2 levels did not significantly affect gene expression. These results inform selection of housekeeping genes for neonatal mRNA research.

Genomic RNA sequence of feline coronavirus strain FCoV C1Je.

This paper reports the first genomic RNA sequence of a field strain feline coronavirus (FCoV). Viral RNA was isolated at post mortem from the jejunum and liver of a cat with feline infectious peritonitis (FIP). A consensus sequence of the jejunum-derived genomic RNA (FCoV C1Je) was determined from overlapping cDNA fragments produced by reverse transcriptase polymerase chain reaction (RT-PCR) amplification. RT-PCR products were sequenced by a reiterative sequencing strategy and the genomic RNA termini were determined using a rapid amplification of cDNA ends PCR strategy. The FCoV C1Je genome was found to be 29,255 nucleotides in length, excluding the poly(A) tail. Comparison of the FCoV C1Je genomic RNA sequence with that of the laboratory strain FCoV FIP virus (FIPV) 79-1146 showed that both viruses have a similar genome organisation and predictions made for the open reading frames and cis-acting elements of the FIPV 79-1146 genome hold true for FCoV C1Je. In addition, the sequence of the 3′-proximal third of the liver derived genomic RNA (FCoV C1Li), which encompasses the structural and accessory protein genes of the virus, was also determined. Comparisons of the enteric (jejunum) and non-enteric (liver) derived viral RNA sequences revealed 100% nucleotide identity, a finding that questions the well accepted ‘internal mutation theory’ of FIPV pathogenicity.

Sustained Expansion of Human Natural Killer Cells for Leukemia Therapy.

Natural killer (NK) cells are a promising tool for cell therapy of hematologic malignancies. They have potential for enhancing graft-versus-leukemia responses in recipients of hematopoietic stem cell transplant, and can also be used in a non-transplant setting, where haploidentical donor NK cells have been shown to expand in vivo. NK cells represent a small subset of peripheral blood cells. Hence, it can be problematic to obtain them in quantities sufficient to exert significant anti-leukemic activity in patients. We sought to identify culture conditions that would stimulate vigorous, sustained and specific expansion of CD56+ CD3− NK cells. NK cell proliferation was stimulated by contact with the K562 leukemia cell line transfected with two NK stimulatory molecules: membrane-bound interleukin 15 and 4-1BB ligand. Exposure of peripheral blood cells from 23 donors to irradiated K562-mb15-41BBL cells in the presence of 10 IU/mL interleukin-2 resulted in a median expansion of CD56+ CD3− cells of 22-fold (range, 9- to 87-fold) after only 7 days of culture; expansion of CD3+ T cells was negligible. After 14 days of culture, K562-mb15-41BBL cells were completely lysed by the NK cells and no further expansion occurred. However, further NK cell expansion could be achieved by addition of fresh K562-mb15-41BBL cells to the cultures. Using this method, NK cell expansions ranged from 2,000- to 98,000-fold (n = 4) after 65 days of culture. We noted that NK cells eventually became unresponsive to stimulation and underwent senescence after 2–5 months of culture. To determine whether NK cell senescence could be overcome by enforced expression of human telomerase reverse transcriptase (hTERT), we stimulated NK cells for 1 week with K562-mb15-41BBL cells and then transfected them using an MSCV retroviral vector and the hTERT gene (gift of Dr. J. Dome, St. Jude). hTERT expression and telomerase activity was demonstrated by reverse transcriptase-polymerase chain reaction and telomerase repeat amplification protocol assay. The cultures were then stimulated with periodic pulses of K562-mb15-41BBL cells. In 2 donors, enforced expression of hTERT overcame senescence: NK cells transfected with an empty vector died after 85 and 170 days of culture, whereas hTERT-NK cells continue to grow after more than 350 days of culture, while retaining a normal karyotype. hTERT-NK cells maintained their cytotoxicity against the NK-sensitive leukemic cell lines K562, KG1, U937, HL60 and Jurkat. They could be also be genetically modified to express anti-CD19 chimeric signaling receptors, thus becoming cytotoxic against NK-resistant CD19+ B-lineage acute lymphoblastic leukemia cells. Cytotoxicity against CD19+ targets was similar to that of NK cells transfected with the signaling receptor after only one week of culture. In conclusion, coculture of human peripheral blood mononuclear cells with pulses of irradiated K562-mb15-41BBL cells allows the generation of a large numbers of NK cells which have powerful anti-leukemic capacity and can be redirected to lyse NK-resistant target cells. Although senescence eventually ensues, this can be overcome by hTERT expression. The culture system described here has now been adapted to large scale expansion for clinical use.

Aberrant nuclear BCL10 expression and lack of t(11;18)(q21;q21) in primary cutaneous marginal zone B-cell lymphoma

Inhibition of apoptosis seems to play an important role in the pathogenesis of marginal zone lymphoma. Apoptosis regulator B-cell lymphoma 10 (BCL10) may show aberrant nuclear localization in some aggressive extracutaneous MALT lymphomas, often in association with a MALT1 gene t(11;18)(q21;q21) translocation. The possible occurrence of this association in primary cutaneous marginal zone lymphoma (PCMZL) remains insufficiently explored. The aim of this study was to evaluate BCL10 protein expression pattern and its possible relationship to the presence of t(11;18)(q21;q21) and other MALT1 gene abnormalities in PCMZL and to assess their clinical significance. The study included 42 consecutive PCMZL patients diagnosed on the basis of the World Health Organization/European Organization for the Research and Treatment of Cancer classification criteria. BCL10 expression was immunohistochemically evaluated in all cases, whereas t(11;18)(q21;q21) reverse transcriptase polymerase chain reaction amplification was performed on 21 samples. In addition, the presence of other MALT1 gene translocations was explored in 26 samples by interphase fluorescence in situ hybridization using a MALT1 locus-specific probe. We observed the presence of aberrant nuclear BCL10 expression in a significant number of PCMZL cases (36%, 15/42). This aberrant expression was significantly related to the development of extracutaneous disease. In contrast, neither the t(11;18)(q21;q21) translocation nor other MALT1 gene translocations could be demonstrated. t(11;18)(q21;q21), strongly linked to extracutaneous MALT lymphomas, does not seem to play a role in PCMZL. The participation of other MALT1 gene translocations in PCMZL pathogenesis seems also unlikely.

Cloning of the Bovine β-Carotene-15,15’-Oxygenase and Expression in Gonadal Tissues

Beta-carotene-15,15'-oxygenase (betaCO), found mainly in intestinal mucosa and liver, is the enzyme responsible for cleaving beta-carotene into retinal, which can be used or stored at these sites or carried by the bloodstream to different target cells within the body. We isolated the cDNA for bovine betaCO and demonstrated its expression in gonadal tissues. A cDNA of 2130 base pairs (bp) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), using degenerate oligonucleotides; the deduced protein shared an identity of 75% with its homologues from other mammalian species. In order to evaluate the expression of this enzyme, we performed RT-PCR and in situ hybridizations in the ovary and testis of bovines. RT-PCR showed the expression of betaCO in testis, ovary, and cultured granulosa cells. In situ hybridization of complete ovary and testis revealed expression in granulosa cells and the corpus luteum in the ovary and in germinal and interstitial cells in the testis. These results suggest that beta-carotene could act as a local source of retinoids, which have been shown to be important during proliferation, differentiation, and maturation of both female and male germinal cells.

Molecular cloning and gonadotropin-releasing hormone regulation of receptor activated for C protein kinase (RACK) in tilapia Oreochromis mossambicus

A receptor for activated C-kinase (RACK) cDNA in the Mozambique tilapia (Oreochromis mossambicus) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and it was compared with other vertebrates. Nucleotide sequence analysis revealed that the full length of cDNA (1109 bp) cloned encompasses 954 bp open reading frame encoding 317 amino acid residues. The deduced amino acid sequence shares 95–99% identity with the teleosts and 77–95% compared with other vertebrates. Like most RACK sequences in other vertebrates, seven repeats of the WD-40 motif are well-conserved in the sequence of the tilapia. Further, the effectiveness of gonadotropin-releasing hormone (GnRH) regulation of RACK LH, and FSH gene expression was further investigated by a single injection of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). The results showed that injection of tilapia brain with either sGnRH or cGnRH-II significantly increased mRNA levels of RACK, which then stimulate LH and FSH. This result suggests that RACK and protein kinase C (PKC) are involved in GnRH-mediated gonadotropin hormone (GtH) release. In addition, cGnRH-II enhanced the FSH and LH mRNA as well as sGnRH expression levels in tilapia; these results further demonstrate that both sGnRH and cGnRH regulate GtH through PKC.

Cloning and expression of rabbit interleukin-15

In order to understand the inflammatory mechanisms related to rabbit interleukin-15 (RIL-15), we cloned and expressed RIL-15 cDNA gene. A cDNA encoding RIL-15 was cloned from heart mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using hIL-15 primers. The RIL-15 cDNA contains an open reading frame (ORF) of 162 amino acids (aa) with a 48 aa leader sequence. The predicted molecular weight of the encoded protein (12.5 kDa) matched the size of recombinant IL-15 on Western blotting in an Escherichia coli (pET32a) expression system. Amino acid and nucleotide sequence analyses of RIL-15 revealed 82.7% and 87% homology with human IL-15 (hIL-15), respectively. RIL-15 is similar to the hIL-15 (hIL-15) in that it contains seven cysteine residues. RT-PCR showed that IL-15 is expressed in many tissues in the rabbit, including heart, spleen, lung, liver, muscle and kidney. Expressed and purified recombinant RIL-15, in the absence of the 48 aa leader sequence, stimulated the proliferation of cells of the mouse T cell line, CTLL-2, and its activity is comparable to hIL-15. Western blotting demonstrated that recombinant RIL-15 can be recognized by anti-IL-15 neutralization antibody. Western blotting also confirmed that IL-15 is present in many tissues including heart, spleen, lung, liver, muscle and kidney.