IDENTIFIKASI IKAN CUPANG (Betta imbelis) TRANSGENIK FOUNDER MEMBAWA GEN PENYANDI HORMON PERTUMBUHAN; Identification of Transgenic Founder Betta Fish (Betta imbelis) Carry Growth Hormone Gene.

Penelitian dilakukan untuk mengidentifikasi keberhasilan introduksi gen penyandi hormon pertumbuhan (Growth Hormone, GH) pada induk F-0 ikan Betta imbellis. Ikan transgenik F-0 dibuat dengan menggunakan metode transfeksi. Identifikasi dilakukan menggunakan metode RT-PCR. RNA total diekstraksi dari embrio pooled sample hasil persilangan induk transgenik dan non-transgenik. Berdasarkan analisis ekspresi gen pada embrio juga menunjukkan adanya aktivitas ekspresi gen GH pada semua perlakuan dibandingkan dengan kontrol (embrio hasil persilangan non-transgenik x non-transgenik). Jumlah individu induk F-0 yang membawa gen GH eksogen berdasarkan analisis PCR dengan DNA template dari sirip ekor adalah sebanyak 16%. Individu positif membawa gen GH eksogen tersebut dibesarkan lebih lanjut untuk memproduksi Betta imbellis transgenik F-1. Kandidat ikan transgenik jantan F-0 dikawinkan dengan ikan non-transgenik betina, sedangkan transgenik F-0 betina dikawinkan dengan non-transgenik jantan. Sebanyak 30-50 butir embrio hasil pemijahan F-0 digabung, kemudian DNA genom diekstrak. Sebagian embrio digunakan untuk ekstraksi RNA total untuk analisis ekspresi mRNA GH eksogen. Hasil analisis PCR menunjukkan bahwa semua sampel embrio dari induk transgenik F-0 dapat terdeteksi gen GH eksogen, sedangkan untuk kontrol (non-transgenik) tidak terdeteksi. Ekspresi mRNA juga terdeteksi pada embrio F-1. Dengan demikian, metode transfeksi embrio Betta imbellis efektif digunakan untuk menghasilkan ikan transgenik, dan sangat berpotensi menghasilkan individu F-1 Betta imbellis dengan pertumbuhan lebih cepat.The study was conducted to identify the successful introduction of the growth hormone gene (Growth Hormone, GH) on the F-0 Betta imbellis broodstock. The F-0 transgenic fish was made through transfection methods. Identification was done using RT-PCR method. Total RNA was extracted from pooled embryos sample. Based on the analysis of gene expression in embryos also showed activity GH gene expression in all treatments compared to the control (non-transgenic x non-transgenic). The number of individuals F-0 which carried exogenous GH gene by PCR analysis of the DNA template of the tail fin was as much as 16%. Positive individuals carried the exogenous GH gene raised further to produce transgenic F-1 B. imbellis. Candidate of transgenic F-0 males fish were mated with non-transgenic female fish, whereas the transgenic F-0 females were mated with non-transgenic males. The 30-50 embryos obtained were combined, then their genomic DNA were extracted. Some of the embryos was used for the extraction of total RNA for analysis of mRNA expression of GH exogenous. The PCR analysis showed that all samples of embryos from the transgenic F-0 broodstock could be detected, whereas for the control (non-transgenic) was not detected. mRNA expression was also detected in embryos of F-1. The average weight of the F-0 broodstocks were 1.55 g and a total length was 12.97 cm. Thus, the transfection methods through betta embryos peaceful effectively generated transgenic fish, and potentially produced fast growth of individuals F-1 Betta imbellis.

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Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: functional implications, molecular mechanisms and signal transduction

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01629 ◽

2005 ◽

Vol 34 (2) ◽

pp. 415-432 ◽

Cited By ~ 30

Author(s):

Hong Zhou ◽

Yonghua Jiang ◽

Wendy K W Ko ◽

Wensheng Li ◽

Anderson O L Wong

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Adenylate Cyclase ◽

Mrna Expression ◽

Grass Carp ◽

Janus Kinase ◽

Growth Hormone Gene ◽

Pituitary Cells ◽

Mrna Levels ◽

Gh Gene

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased ‘steady-state’ GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44MAPK and P38 MAPK. These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.

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Reverse Transcription-PCR Analysis of the Regulation of the Manganese Peroxidase Gene Family

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.569-574.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 569-574 ◽

Cited By ~ 60

Author(s):

Jessica M. Gettemy ◽

Biao Ma ◽

Margaret Alic ◽

Michael H. Gold

Keyword(s):

Gene Expression ◽

Reverse Transcription ◽

Manganese Peroxidase ◽

Transcript Level ◽

Pcr Analysis ◽

Rt Pcr ◽

Total Rna ◽

Nitrogen Levels ◽

Reverse Transcription Pcr ◽

Low Levels

ABSTRACT Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequencedP. chrysosporium mnp genes, i.e., mnp1,mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 μM Mn, the levels of the mnp1 and mnp2 transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of themnp3 transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the mnp1 transcript was present at relatively low levels on days 3 through 7, the mnp2transcript level peaked on day 5 and the mnp3 transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures, mnp2 was the major expressed mnp gene, whereas in large agitated cultures,mnp1 was the major expressed mnp gene.

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PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00188.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. R1625-R1633 ◽

Cited By ~ 18

Author(s):

Christian Klausen ◽

Takeshi Tsuchiya ◽

John P. Chang ◽

Hamid R. Habibi

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Primary Cultures ◽

Gonadotropin Releasing Hormone ◽

Growth Hormone Gene ◽

Pituitary Cells ◽

Mrna Levels ◽

Releasing Hormone ◽

Gh Secretion ◽

Gh Gene

Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and stimulates the synthesis and secretion of gonadotropin hormones. In addition, GnRH also stimulates the production and secretion of growth hormone (GH) in some fish species and in humans with certain clinical disorders. In the goldfish pituitary, GH secretion and gene expression are regulated by two endogenous forms of GnRH known as salmon GnRH and chicken GnRH-II. It is well established that PKC mediates GnRH-stimulated GH secretion in the goldfish pituitary. In contrast, the signal transduction of GnRH-induced GH gene expression has not been elucidated in any model system. In this study, we demonstrate, for the first time, the presence of novel and atypical PKC isoforms in the pituitary of a fish. Moreover, our results indicate that conventional PKCα is present selectively in GH-producing cells. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators (phorbol ester or diacylglycerol analog) did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (calphostin C and GF109203X) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in the goldfish pituitary. Instead, PD98059 inhibited GnRH-induced GH gene expression, suggesting that the ERK signaling pathway is involved. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.

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Coho salmon (Oncorhynchus kisutch) transgenic for a growth hormone gene construct exhibit increased rates of muscle hyperplasia and detectable levels of differential gene expression

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f00-015 ◽

2000 ◽

Vol 57 (5) ◽

pp. 939-950 ◽

Cited By ~ 50

Author(s):

James A Hill ◽

Anders Kiessling ◽

Robert H Devlin

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Differential Gene Expression ◽

Coho Salmon ◽

Oncorhynchus Kisutch ◽

Transgenic Fish ◽

Growth Hormone Gene ◽

Gene Construct ◽

Muscle Hyperplasia ◽

Differential Gene

Transgenic coho salmon (Oncorhynchus kisutch) containing a growth hormone gene construct were compared with nontransgenic coho salmon in terms of gross anatomy, muscle cellularity, muscle enzyme activity, and differential gene expression. Transgenic fish were found to have significantly higher numbers of small-diameter muscle fibres in both the dorsal and lateral region of the somitic muscle, suggesting that they grow by greater rates of hyperplasia relative to slower growing nontransgenic fish. Higher levels of activity were found for phosphofructokinase and cytochrome oxidase in white muscle of the transgenic fish. This difference indicates a higher glycolytic and aerobic requirement in the muscle of transgenic fish. Subtractive hybridisation of muscle RNA of transgenic fish from control fish provided a library of cDNAs whose expression is upregulated in the transgenic fish. This library contains genes that may be involved in, or related to, both high growth rates and muscle hyperplasia. We have sequenced a number of fragments and have found a preponderance of myosin light chain 2 mRNAs, consistent with a putative high level of expression in the early stages of muscle fibre construction.

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Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00298.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. H1625-H1631 ◽

Cited By ~ 37

Author(s):

Katherine L. Tran ◽

Xiangru Lu ◽

Ming Lei ◽

Qingping Feng ◽

Qingyu Wu

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Mrna Expression ◽

Rat Model ◽

Left Ventricular ◽

Biologically Active ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Neonatal Cardiomyocytes

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells ( r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.

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IDENTIFICATION OF GROWTH HORMONE GENE OF Pangasionodon hypophthalmus

Indonesian Aquaculture Journal ◽

10.15578/iaj.4.1.2009.9-17 ◽

2009 ◽

Vol 4 (1) ◽

pp. 9

Author(s):

Raden Roro Sri Pudji Sinarni Dewi ◽

Agus Oman Sudrajat ◽

Alimuddin Alimuddin ◽

Komar Sumantadinata

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Rna Extraction ◽

Pcr Amplification ◽

Transgenic Fish ◽

Nucleotide Sequencing ◽

Tryptophan Residue ◽

Growth Hormone Gene ◽

Reading Frame ◽

Gh Gene

Identification of growth hormone (GH) gene in a target fish is the first step in the construction of “all fish genes transfer vector” to generate transgenic fish. The research was done to identify and characterize the GH gene of Pangasionodon hypophthalmus. There were several activities performed in identifying the GH gene: RNA extraction, cDNA synthesis, PCR amplification, and DNA fragment isolation. The characterizations were done using the nucleotide sequencing engine ABIPRISM 3100. The results were then analyzed using BLASTN/P and GENETYX version 7 program. The full-length GH gene of P. hypophthalmus was 1151 bp in length, coding for an open reading frame (ORF) of 603 bp. The 5’ and 3’ untranslated regions of the GH gene were 22 bp and 526 bp long, respectively. The GH gene of P. hypophthalmus had some common characteristics that are owned by GH genes, such as single tryptophan residue (W) on the 104th amino acid, 5 cysteine residues (C) on the amino acid 71, 135, 173, 190, and 198 and a motif of Asn-Xaa-Thr on C terminus which is the potential location for N-linked glycosilation. Polyadenylation signal (aataaa) was on the 14 bp at the upstream of polyadenylation location. Growth hormone of P. hypophthalmus consisted of over 200 amino acids from GH cDNA deduction. The highest proportion of amino acid composition was leusin (14%) while the lowest was tryptophan (0.5%).

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Genomic structure and sequence of the gilthead seabream (Sparus aurata) growth hormone-encoding gene: Identification of minisatellite polymorphism in intron I

Genome ◽

10.1139/g00-051 ◽

2000 ◽

Vol 43 (5) ◽

pp. 836-845 ◽

Cited By ~ 39

Author(s):

R Almuly ◽

B Cavari ◽

H Ferstman ◽

O Kolodny ◽

B Funkenstein

Keyword(s):

Growth Hormone ◽

Tandem Repeats ◽

Sparus Aurata ◽

Genomic Structure ◽

Pcr Analysis ◽

Gilthead Seabream ◽

Growth Hormone Gene ◽

Response Element ◽

First Intron ◽

Gh Gene

The growth hormone (GH) gene of the gilthead seabream (Sparus aurata) (saGH) has been cloned, sequenced, and characterized. The saGH gene spans approximately 4.3 kb and consists of six exons and five introns, as found for all cloned teleost GH genes with the exception of carps and catfish. The first and third introns contain long stretches of repetitive tandem repeats. The second intron, which is unusually long compared with that in other teleosts (and other vertebrates) spans 1747 nucleotides (nt) and contains several inverted repeats. Intron-targeted polymerase chain reaction (PCR) analysis identified length polymorphism of the first intron. Sequence analysis of four variants (405, 424, 636, and 720 nt) out of many variants found revealed that the variation in length is due to differences in the number of repeat monomers (17-mer or 15-mer) as well as minor changes in their length. This repeat unit contains the consensus half-site motif of the thyroid hormone response element (TRE) and estrogen response element (ERE). Polymorphism was found also in the third intron. This is the first report of such high polymorphism of the first intron of GH gene in a vertebrate.Key words: growth hormone, gene, intron polymorphism, fish, Sparus aurata.

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Developmental Changes of Growth Hormone Mrna Abundance in Pituitary Tissue of Jinhua and Landrace Gilts

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.343-344.412 ◽

2011 ◽

Vol 343-344 ◽

pp. 412-416

Author(s):

Zhi Guo Miao ◽

Guo Wang Li ◽

Shi Zhu Wang ◽

Xin Yao Chang ◽

Hong Bing Xie ◽

...

Keyword(s):

Growth Hormone ◽

Mrna Expression ◽

Age Groups ◽

Rt Pcr ◽

Developmental Patterns ◽

Expression Levels ◽

Pituitary Tissue ◽

Breed Differences ◽

Gh Gene ◽

Mrna Expression Levels

In this pepar we investigated the developmental patterns of expression of growth hormone (GH) gene in pituitary tissue in pigs of different breeds and their effects on the carcass fat contents. 3 Jinhua gilts and 3 Landrace gilts were sampled at 35, 80 and 125 days of age, respectively. Carcass fat contents were determined. Pituitary tissue was sampled and total RAN was extracted to determine GH mRNA expression levels by semi-quantitative RT-PCR. The results showed that the contents of carcass fat increased with growth and showed significant differences (P﹤0.05) between different age groups in the two breeds. Furthermore, carcass fat contents in Jinhua gilts were higher than that in Landrace gilts during growth (P﹤0.05). GH mRNA expression levels decreased with age and displayed breed differences. Jinhua gilts showed lower abundance of GH mRNA compared with Landrace gilts at 35, 80 and 125 days of age (P﹤0.05). In addition, GH mRNA expression level was negatively related to carcass fat content in Jinhua and Landrace gilts (r = -0.790 (P = 0.01), r = -0.755 (P = 0.02), respectively).

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