scholarly journals Molecular cloning and mRNA expression analysis of two GH secretagogue receptor transcripts in orange-spotted grouper (Epinephelus coioides)

2008 ◽  
Vol 199 (2) ◽  
pp. 253-265 ◽  
Author(s):  
Ting Chen ◽  
Zhiguo Tang ◽  
Aifen Yan ◽  
Wensheng Li ◽  
Haoran Lin
Keyword(s):  
Amino Acids ◽  
Pituitary Gland ◽  
Feedback Regulation ◽  
Expression Level ◽  
Epinephelus Coioides ◽  
Reading Frame ◽  
Mrna Expression Analysis ◽  
Acyl Ghrelin

GH secretagogue receptor (GHSR) is the receptor of ghrelin, a circulating GH-releasing and appetite-inducing hormone. In this paper, two Ghsr cDNAs, gpGhsr1a and gpGhsr1b, were identified and characterized in a teleost, the orange-spotted grouper (Epinephelus coioides). The gpGHSR1a is 1512 bp in length with an open reading frame (ORF) that encodes a protein of 383 amino acids with seven transmembrane (TM) domains, while the 1703 bp gpGHSR1b contains an ORF encoding for 303 amino acids with five TM domains. Comparison between cDNA and gene sequences showed that the two transcripts are two alternative splicing forms of a single gpGhsr gene. Tissue distribution and ontogeny of two gpGhsr mRNAs were examined by RT-PCR. The gpGHSR1a is mainly expressed in brain and pituitary gland, when compared with a more widespread expression of gpGHSR1b. During embryonic and larval development, the gpGhsr1b mRNA appears before the gpGhsr1a mRNA. Furthermore, quantitative real-time PCR performed on brain showed that both transcripts have the highest expression level in the pituitary gland. The expression level of gpGHSR1a was generally higher than that of gpGHSR1b. GHSR expressing cells were also detected widely in grouper brain by in situ hybridization, with a broader distribution than previous reports in mammals. Finally, an in vitro study showed that expression of both gpGHSR transcripts in pituitary and hypothalamus is downregulated by GH and ghrelin but not by des-acyl ghrelin, and this suggests that feedback regulation of GHSR also exists in teleostean fishes.

scholarly journals Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

2001 ◽  
Vol 21 (1) ◽  
pp. 354-366 ◽  
Author(s):  
Carolina Sousa ◽  
Christina Johansson ◽  
Celine Charon ◽  
Hamid Manyani ◽  
Christof Sautter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Rna Structure ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Cortical Cells ◽  
Root Cortex ◽  
Reading Frame ◽  
Early Nodulin

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

scholarly journals Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

1995 ◽  
Vol 6 (2) ◽  
pp. 171-183 ◽  
Author(s):  
H Yu ◽  
C V Nicchitta ◽  
J Kumar ◽  
M Becker ◽  
I Toyoshima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Hydrophobic Region ◽  
Reading Frame

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

scholarly journals Primary sequence and developmental expression of a novel Drosophila melanogaster src gene.

1987 ◽  
Vol 7 (6) ◽  
pp. 2119-2127 ◽  
Author(s):  
R J Gregory ◽  
K L Kammermeyer ◽  
W S Vincent ◽  
S G Wadsworth
Keyword(s):  
Drosophila Melanogaster ◽  
Blot Hybridization ◽  
Structural Features ◽  
Developmental Expression ◽  
Reading Frame ◽  
Adult Females ◽  
Src Gene ◽  
Free Translation

We have sequenced a cDNA clone for the Drosophila melanogaster gene Dsrc28C, a homolog of the vertebrate gene c-src. The cDNA contains a single open reading frame encoding a protein of 66 kilodaltons which contains features highly conserved within the src family of tyrosine protein kinases. Novel structural features of the Dsrc28C protein include a basic pI and a polyglycine domain near the amino terminus. Cell-free translation of in vitro-transcribed RNA yielded a protein of the predicted size which could be immunoprecipitated by anti-v-src antisera. RNA blot hybridization revealed that the gene is expressed predominantly during embryogenesis, in imaginal disks of third-instar larvae, and in adult females. In situ hybridization showed that expression in adult females is largely confined to nurse cells and developing oocytes.

scholarly journals Rumen degradability and ileal digestibility of proteins and amino acids of feedstuffs for cows

2014 ◽  
Vol 83 (3) ◽  
pp. 225-231
Author(s):  
Iveta Maskaľová ◽  
Vladimír Vajda ◽  
Marek Krempaský ◽  
Lukáš Bujňák
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Crude Protein ◽  
Amino Acid Profile ◽  
Ileal Digestibility ◽  
Corn Gluten ◽  
Corn Gluten Feed ◽  
Gluten Feed

Knowledge of the profile of amino acids of the rumen-undegradable protein can help in the formulation of diets to provide amino acids that complement microbial protein as well as supply amino acids, which are most limiting for milk production. Three non-lactating cows fitted with rumen cannulas were used to determine the effect ofin siturumen degradation on crude protein and amino acid profile of rumen-undegraded protein of feedstuffs. The obtained values of rumen degradability of crude protein with significant difference (P< 0.001) between feeds ranged from 20.3 to 76.3% (mean 62.0 ± 17.9%) and values of total amino acids ranged from 30.9% in corn gluten meal to 83.8% in corn gluten feed (mean 67.5 ± 16.4%). Anin vitromodified 3-step method was used to determine intestinal digestibility. Intestinal digestibility of undegraded protein varied from 54.5 ± 1.4% in raw soybean to 95.2 ± 1.0% in corn gluten feed. The absorbable amino acid profile of rumen-undegraded protein for each feedstuff was compared with profiles of the original feedstuff and the rumen-exposed undegraded protein. Absorbable lysine (9.3 ± 1.1 g/kg of crude protein) was higher in products of soybean and sunflower cake. Corn gluten feed and meal supplied more absorbable methionine (3.6 ± 0.6 g/kg of crude protein). This study showed that the digestibility factor of crude protein and amino acid based onin situandin vitromethods for thermal treatment of protein feeds can be used in models to optimize the amino acid nutrition of dairy cows and expand knowledge about rumen degradability and ileal digestibility of amino acids in feedstuffs.

scholarly journals A C-terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein

2001 ◽  
Vol 1 (3) ◽  
pp. 122-128 ◽  
Author(s):  
Li Xiaofang ◽  
Mohammad Zafrullah ◽  
Faizan Ahmad ◽  
Shahid Jameel
Keyword(s):  
Amino Acids ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Acute Form ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressedin vitroor in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.

scholarly journals Fusion of the N-terminal 119 amino acids with the RelA-CTD renders its growth inhibitory effects (p)ppGpp-dependent

2021 ◽  
Author(s):  
Jayashree Pohnerkar ◽  
Krishma Tailor ◽  
Prarthi Sagar ◽  
Keyur Dave
Keyword(s):  
Amino Acids ◽  
Feedback Regulation ◽  
Inhibitory Effects ◽  
E Coli ◽  
Growth Inhibitory ◽  
In The Wild ◽  
Regulatory Aspect ◽  
And Function

The guanosine nucleotide derivatives ppGpp and pppGpp are central to the remarkable capacity of bacteria to adapt to fluctuating environment and metabolic perturbations. These alarmones are synthesized by two proteins, RelA and SpoT in E. coli and the activities of each of the two enzymes are highly regulated for homeostatic control of (p)ppGpp levels in the cell. Although the domain structure and function of RelA are well defined, the findings of this study unfold the regulatory aspect of RelA that is possibly relevant in vivo. We uncover here the importance of the N-terminal 1-119 amino acids of the enzymatically compromised (p)ppGpp hydrolytic domain (HD) of monofunctional RelA for the (p)ppGpp mediated regulation of RelA-CTD function. We find that even moderate level expression of RelA appreciably reduces growth when the basal levels of (p)ppGpp in the cells are higher than in the wild type, an effect independent of its ability to synthesize (p)ppGpp. This is evidenced by the growth inhibitory effects of oversynthesis of the RelA-CTD in the relA+ strain but not in relA null mutant, suggesting the requirement of the functional RelA protein for basal level synthesis of (p)ppGpp, accordingly corroborated by the restoration of the growth inhibitory effects of the RelA-CTD expression in the relA1 spoT202 mutant. The N-terminal 119 amino acids of RelA fused in-frame with the RelA-CTD, both from 406-744 amino acids (including TGS) and from 454-744 amino acids (sans TGS) caused growth inhibition only in spoT1 and spoT202 relA1 mutants, uncovering the hitherto unrealized (p)ppGpp-dependent regulation of RelA-CTD function. An incremental rise in the (p)ppGpp levels is proposed to progressively modulate the interaction of RelA-CTD with the ribosomes, with possible implications in the feedback regulation of the N-terminal (p)ppGpp synthesis function, a proposal that best explains the nonlinear relationship between (p)ppGpp synthesis and increased ratio of RelA:ribosomes, both in vitro as well as in vivo.

scholarly journals Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

2020 ◽  
Author(s):  
Werner J. D. Ouwendijk ◽  
Daniel P. Depledge ◽  
Labchan Rajbhandari ◽  
Tihana Lenac Rovis ◽  
Stipan Jonjic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Varicella Zoster Virus ◽  
Viral Gene Expression ◽  
Fusion Transcript ◽  
Viral Gene ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Rapid Amplification

SummaryVaricella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5’ rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

Rumen degradation and intestinal digestibility of rumen protected amino acids: comparison between in situ and in vitro data

2003 ◽  
Vol 108 (1-4) ◽  
pp. 223-229 ◽  
Author(s):  
Filippo Rossi ◽  
Moschini Maurizio ◽  
Masoero Francesco ◽  
Cavanna Giovanna ◽  
Piva Gianfranco
Keyword(s):  
Amino Acids ◽  
Rumen Degradation ◽  
Vitro Data ◽  
In Vitro Data ◽  
Intestinal Digestibility

scholarly journals A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site.

1992 ◽  
Vol 12 (2) ◽  
pp. 685-695 ◽  
Author(s):  
V Bours ◽  
P R Burd ◽  
K Brown ◽  
J Villalobos ◽  
S Park ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Peripheral Blood ◽  
Carcinoma Cells ◽  
Nf Kappa B ◽  
Reading Frame ◽  
Amino Terminal ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Inducible Gene

A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors.

scholarly journals Role of the UL25 Protein in Herpes Simplex Virus DNA Encapsidation

2008 ◽  
Vol 83 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Shelley K. Cockrell ◽  
Minerva E. Sanchez ◽  
Angela Erazo ◽  
Fred L. Homa
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Cleavage ◽  
Virus Protein ◽  
Viral Dna ◽  
Reading Frame ◽  
Viral Genomes ◽  
Simplex Virus

ABSTRACT The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.

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