scholarly journals Identification and tissue distribution of mRNAs encoding salmon-type calcitonins-IV and -V in the rainbow trout

2004 ◽  
Vol 32 (3) ◽  
pp. 963-974 ◽  
Author(s):  
Y Hidaka ◽  
M Suzuki
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Tissue Distribution ◽  
Restriction Enzymes ◽  
Tissue Expression ◽  
Pcr Analysis ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Parenchymal Cells ◽  
Ultimobranchial Gland

Four types of calcitonin are produced in salmonid fish, although their functional diversity is almost unknown. To explore the significance of these isoforms, we have characterized salmon-type calcitonin (sCT) mRNAs in the rainbow trout (Oncorhynchus mykiss), and examined their tissue distribution. In addition to the previously isolated sCT-I cDNAs, two new forms of sCT cDNA were cloned from the ultimobranchial gland, and one of them (sCT-IV cDNA) was predicted to encode an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-IV, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. The sCT-IV precursor was 78% identical with the rainbow trout sCT-I precursors. The other cloned cDNA encoded a precursor for a novel CT, sCT-V. The sCT-V peptide was different from sCT-IV by only one amino acid residue: Val at position 8 in the latter was replaced by Met. The sCT-V precursor had 80 and 90% identity with the sCT-I and -IV precursors respectively. No cDNA clones were obtained for sCTs-II or -III.Tissue distribution of sCT-I, -IV and -V mRNAs was examined by RT-PCR and specific cleavage with restriction enzymes. An amplified fragment from sCT-I mRNA was detected not only in the ultimobranchial gland, but also in the gills, testis and ovary. RT-PCR analysis coupled to restriction digestion further revealed that sCT-IV mRNA was expressed in both the testis and the ultimobranchial gland. The expression sites of sCT-IV mRNA were localized to the Leydig cells of the testis and to the parenchymal cells of the ultimobranchial gland, by in situ hybridization histochemistry. Although the amino acid sequence of sCT-V peptide was nearly the same as that of sCT-IV, the sCT-V gene showed a much wider pattern of expression: the band amplified by RT-PCR was detected in all the tissues examined except the kidney, gills and blood cells. The sCT-V mRNA was shown to be localized in the parenchymal cells of the ultimobranchial gland, but not in other tissues at the cellular level, suggesting very low expression of sCT-V mRNA in those tissues. Our results show different patterns of tissue expression of three types of sCT genes in the rainbow trout, suggesting that sCTs-I, -IV and -V might differ in their local actions.

Cloning of rabbit prolactin cDNA and prolactin gene expression in the rabbit mammary gland

1996 ◽  
Vol 16 (1) ◽  
pp. 27-37 ◽  
Author(s):  
L Gabou ◽  
M Boisnard ◽  
I Gourdou ◽  
H Jammes ◽  
J-P Dulor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Amino Acid ◽  
Untranslated Regions ◽  
Pcr Analysis ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Prolactin Gene ◽  
New Zealand White Rabbits ◽  
Rna Probe

ABSTRACT cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5′ and 3′ untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93–78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

scholarly journals Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes

2006 ◽  
Vol 190 (3) ◽  
pp. 879-888 ◽  
Author(s):  
Dilip K Garikipati ◽  
Scott A Gahr ◽  
Buel D Rodgers
Keyword(s):  
Rainbow Trout ◽  
Genomic Organization ◽  
Expression Profiles ◽  
Muscle Growth ◽  
Fish Tissue ◽  
Tissue Expression ◽  
Negative Regulator ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Quantitative Expression

Myostatin is a potent negative regulator of skeletal muscle growth. Although several cDNA clones have been characterized in different vertebrates, the genomic organization and bioactivity of non-mammalian homologs have not. The intron/exon organization and promoter subsequence analysis of two rainbow trout myostatin genes, rtMSTN-1a and rtMSTN-1b (formerly 1 and 2 respectively), as well as a quantitative assessment of their embryonic, larval, and adult tissue expression profiles are reported herein. Each gene was similarly organized into three exons of 490, 368, and 1600 bp for MSTN-1a and 486, 386, and 1419 bp for MSTN-1b. Comparative mapping of coding regions from several vertebrate myostatin genes revealed a common organization between species, including conserved pre-mRNA splice sites; the first among the fishes and the second across all vertebrate species. In silico subsequence analysis of the promoter regions identified E-boxes and other putative myogenic response elements. However, the number and diversity of elements were considerably less than those found in mammalian promoters or in the recently characterized zebrafish MSTN-2 gene. A quantitative analysis of the embryonic expression profile for both genes indicates that rtMSTN-1a expression is consistently greater than that of rtMSTN-1b and neither gene is significantly expressed throughout gastrulation. Expression of both steadily increases fourfold during somitogenesis and subsides as this period ends. After eyeing, however, rtMSTN-1a mRNA levels ultimately rise 20-fold by day 49 and peak before hatching and yolk sac absorption (YSA). Levels of rtMSTN-1b rise similarly, but do not peak before YSA. An analysis of adult (2-year-old fish) tissue expression indicates that both transcripts are present in most tissues although levels are highest in brain, testes, eyes, muscle, and surprisingly spleen. These studies suggest that strong selective pressures have preserved the genomic organization of myostatin genes throughout evolution. However, the different expression profiles and putative promoter elements in fishes versus mammals suggests that limitations in myostatin function may have evolved recently.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Ancient and recent duplications of the rainbow trout Wilms' tumor gene

Genome ◽  
2001 ◽  
Vol 44 (3) ◽  
pp. 455-462 ◽  
Author(s):  
Joseph P Brunelli ◽  
Barrie D Robison ◽  
Gary H Thorgaard
Keyword(s):  
Rainbow Trout ◽  
Tumor Suppressor ◽  
Wilms Tumor ◽  
Gene Families ◽  
Duplication Event ◽  
Tissue Expression ◽  
Wt1 Gene ◽  
Rt Pcr ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Bony Fishes

The Wilms' tumor suppressor (WT1) gene plays an important role in the development and functioning of the genitourinary system, and mutations in this gene are associated with nephroblastoma formation in humans. Rainbow trout (Oncorhynchus mykiss) is one of the rare animal models that readily form nephroblastomas, yet trout express three distinct WT1 genes, one of which is duplicated and inherited tetrasomically. Sequence analyses suggest an ancient gene duplication in the common ancestor of bony fishes resulted in the formation of two WT1 gene families, that conserve the splicing variations of tetrapod WT1, and a second duplication event occurred in the trout lineage. The WT1 genes of one family map to linkage groups 6 and 27 in the trout genome map. Reverse transcribed polymerase chain reaction (RT-PCR) expression analysis demonstrated little difference in WT1 tissue expression pattern between genes.Key words: tumor suppressor, nephroblastoma, RT-PCR expression, kidney, cancer, cDNA, gene mapping.

scholarly journals Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA

1992 ◽  
Vol 285 (1) ◽  
pp. 17-23 ◽  
Author(s):  
S Hübner ◽  
F Michel ◽  
V Rudloff ◽  
H Appelhans
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Untranslated Region ◽  
Band 3 ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Band 3 Protein ◽  
Lower Vertebrate ◽  
Calculated Molecular Mass

In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5′-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3′-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins.

Bioavailability and tissue distribution of amino acid-chelated trace elements in rainbow trout Oncorhynchus mykiss

2003 ◽  
Vol 69 (4) ◽  
pp. 722-730 ◽  
Author(s):  
Mary Jane S APINES ◽  
Shuichi SATOH ◽  
Viswanath KIRON ◽  
Takeshi WATANABE ◽  
Shoji FUJITA
Keyword(s):  
Trace Elements ◽  
Rainbow Trout ◽  
Amino Acid ◽  
Oncorhynchus Mykiss ◽  
Tissue Distribution ◽  
Rainbow Trout Oncorhynchus Mykiss

scholarly journals First Report of Passiflora chlorosis virus in Bituminaria bituminosa in Europe

Plant Disease ◽  
2009 ◽  
Vol 93 (2) ◽  
pp. 196-196 ◽  
Author(s):  
L. Cardin ◽  
B. Moury
Keyword(s):  
Amino Acid ◽  
Green Peach Aphid ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Coding Region ◽  
Rt Pcr ◽  
Leaf Extracts ◽  
Chenopodium Amaranticolor ◽  
First Report ◽  
Bituminaria Bituminosa

Bituminaria bituminosa (L.) Stirton (pitch trefoil) is a perennial legume endemic to the Mediterranean Basin used as forage in arid areas and for stabilization of degraded soils. Mosaic and chlorotic ringspot symptoms have been observed in leaves of B. bituminosa in the Provence-Alpes-Côte d'Azur and Rhône-Alpes regions (France), Liguria (Italy), and Spain since 1975. In crude leaf extracts from more than 50 samples of diverse geographical origins, flexuous particles 680 to 720 nm long and 12 nm wide and pinwheel-like inclusions have been observed with the electron microscope, suggesting infection with a member of the family Potyviridae. The presence of a virus was confirmed by the use of potyvirus-polyvalent ELISA reagents (Potyvirus group test; Agdia, Elkhart, IN) and by the amplification of a DNA fragment of the expected size (≈1,650 bp) with extracts of isolates from different locations using reverse transcription (RT)-PCR with primers specific to members of the Potyviridae (3) corresponding to the 3′ end of the virus genome. The amplified fragment of an isolate from Coaraze (Alpes Maritimes Department, France) was cloned and two cDNA clones corresponding to this amplicon were sequenced (GenBank Accession Nos. EU334546 and EU334547). These two sequences facilitated development of new primers (5′-AAARGCRCCCTATATAGCAG-3′ and 5′-TATAAAGGTAACGCTAGGTGG-3′) to specifically amplify and sequence the coat protein (CP)-coding region of isolates of the virus from five additional French locations. The amino acid sequences of the CP amplicon were more than 96% identical among the French isolates. Comparison with other virus sequences with the BLASTn program revealed that these isolates belonged to the same species as the potyvirus Passiflora chlorosis virus (2), with 89 to 90% and 95 to 97% identity at the nucleotide and amino acid levels, respectively, for the CP-coding region (1). The host range of the virus was evaluated by manual inoculation with the Coaraze isolate and was found to be very narrow. No symptoms and no infections were obtained in Capsella bursa-pastoris, Capsicum annuum, Claytonia perfoliata, Cucumis melo, Cucumis sativus, Cucurbita pepo, Datura stramonium, Gomphrena globosa, Medicago sativa, Nicotiana benthamiana, N. glutinosa, N. tabacum, Ocimum basilicum, Petunia hybrida, Phaseolus mungo, Physalis peruviana, Pisum sativum, Psoralea glandulosa, Ranunculus sardous, Salvia splendens, Solanum lycopersicum, Trifolium repens, Vicia faba, Vigna unguiculata, or Zinnia elegans. Necrotic local lesions were observed in Chenopodium amaranticolor, C. quinoa, and in all eight cultivars of Phaseolus vulgaris tested. The virus was transmitted either manually or by the green peach aphid (Myzus persicae) to healthy B. bituminosa seedlings. Symptoms appeared in 10 to 15 weeks, and the virus was detected in the symptomatic plants by RT-PCR. To our knowledge, this is the first report of a virus infecting B. bituminosa. References: (1) M. J. Adams et al. Arch. Virol. 150:459, 2005. (2) C. A. Baker and L. Jones. Plant Dis. 91:227, 2007. (3) A. Gibbs and A. M. Mackenzie. J. Virol. Methods 63:9, 1997.

Structures and expression patterns of two cDNA clones encoding S-adenosyl-L-methionine synthetase from the root nodule of Elaeagnus umbellata

2001 ◽  
Vol 28 (9) ◽  
pp. 951
Author(s):  
Sang Ho Lee ◽  
Ho Bang Kim ◽  
Chung Sun An
Keyword(s):  
Amino Acid ◽  
Expression Pattern ◽  
Root Nodule ◽  
Vascular System ◽  
Expression Patterns ◽  
Amino Acid Level ◽  
Nodule Development ◽  
Pcr Analysis ◽  
Cdna Clones ◽  
Elaeagnus Umbellata

This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Two cDNA clones encoding S-adenosyl-L-methionine synthetase (SAMS) were isolated from the root nodule cDNA library of Elaeagnus umbellata Thunberg and analysed on the basis of deduced amino acid sequence and expression pattern. Two EuSAMS clones shared 75–84% identity at the nucleotide level, and 85–95% identity at the amino acid level, with the other plant SAMS genes. Genomic Southern hybridization revealed the presence of more than two copies of SAMS genes in the genome of E. umbellata. Reverse transcriptase-mediated polymerase chain reaction (RT–PCR) analysis showed EuSAMS1 transcripts were more abundant than those of EuSAMS2. Similar to the expression pattern of other plant SAMS genes, both genes were expressed at higher levels in root than in leaf. During nodule development, expression of both genes was increased, with the highest level at 6–8 week after inoculation, and decreased rapidly thereafter. In situ hybridization analysis also showed both SAMS transcripts in the meristem zone, the infected cells of the fixation zone and in the central vascular system of root nodules. However, EuSAMS2 transcripts were strongly detected in the prefixation zone, whereas EuSAMS1 transcripts were hardly detected. These results suggest different regulatory mechanisms for the two genes in the root nodule. The expression pattern of SAMS genes in the root nodule may correlate mostly with cell wall synthesis, polyamine biosynthesis and other methylation-mediated functions.

scholarly journals Identification of the gene for disaggregatase fromMethanosarcina mazei

Archaea ◽  
2008 ◽  
Vol 2 (3) ◽  
pp. 185-191 ◽  
Author(s):  
Naoki Osumi ◽  
Yoshihiro Kakehashi ◽  
Shiho Matsumoto ◽  
Kazunari Nagaoka ◽  
Junichi Sakai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Cells ◽  
Pcr Analysis ◽  
Amino Acid Residues ◽  
Wall Function ◽  
Rt Pcr ◽  
Methanosarcina Mazei ◽  
C Terminus ◽  
First Time

The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates ofMethanosarcina mazeito single cells, were determined for three strains ofM. mazei(S-6T, LYC and TMA). Thedaggenes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genusMethanosarcinaand may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed inEscherichia coliand preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed thatdagwas transcribed to mRNA inM. mazeiLYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change ofMethanosarcinaspp. from aggregate to single cells has been identified.

scholarly journals The Cryptococcus neoformans GeneDHA1 Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice

2000 ◽  
Vol 68 (11) ◽  
pp. 6196-6201 ◽  
Author(s):  
M. Alejandra Mandel ◽  
Greg G. Grace ◽  
Kris I. Orsborn ◽  
Fredda Schafer ◽  
Juneann W. Murphy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cryptococcus Neoformans ◽  
Culture Filtrate ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Delayed Type Hypersensitivity ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Fruiting Body Formation

ABSTRACT When mice are vaccinated with a culture filtrate fromCryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-lengthDHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5′ rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes(shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded byDHA1. Recombinant DHA1 protein expressed inEscherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.

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