Red Fluorescent Protein Expression in Transgenic Founder of Angelfish (Pterophyllum sp) Driven by Zebrafish Myosin Light Chain 2 Promoter

Angelfish (Pterophyllum sp.) are attractive fish popular with aquarists. The introduction of fluorescent protein genes into angelfish has been reported, but specific techniques have not been revealed. This study aimed to develop a strategy to produce red fluorescent protein (RFP) transgenic angelfish driven by the myosin light chain 2 (mylz2) promoter from zebrafish. A 1999 bp Mylz2 promoter fragment was isolated from zebrafish muscle genomic DNA. This promoter fragment was then cloned into the plasmid pDsred2-1 open-loop at restriction enzyme SacI and AgeI sites to create the final transgene construct pMylz2-RFP. Angelfish embryos at one cell stage were microinjected with approximately 100 pg of the plasmid pMylz2-RFP. From 524 microinjected embryos, 16 successfully hatched, while 12 showed red fluorescence signals. Two larvae survived to 2 months of age. They showed significant red fluorescence expression in the muscles, suggesting that the angelfish could be used as potential transgenic founders to evaluate the next generation of stable red fluorescence expression transgenic fish.

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. Our simulations identified a possible mechanism that explains life-long maintenance of ASE neuron fate in C. elegans by the terminal selector transcription factor CHE-1. Here, fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensures continued che-1 expression by preferentially binding the che-1 promoter. We provide experimental evidence for this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems.

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

SummaryCell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. We uncovered a mechanism that explains life-long maintenance of ASE neuron fate in C. elegans by the terminal selector transcription factor CHE-1. Fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensure continued che-1 expression by preferentially binding the che-1 promoter. We validated this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems.

Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa

Root-specific promoters are useful in plant genetic engineering, primarily to improve water and nutrient absorption. The aim of this study was to clone and characterise the promoter of the Oryza sativa L. alkenal reductase (OsAER1) gene encoding 2-alkenal reductase, an NADPH-dependent oxidoreductase. Expression analysis using quantitative real-time PCR confirmed the root-specific expression of the OsAER1 gene. Subsequently, a 3082-bp fragment of the OsAER1 promoter was isolated from a local Indonesian rice cultivar, Awan Kuning. Sequencing and further nucleotide sequence analysis of the 3082-bp promoter fragment (PA-5) revealed the presence of at least 10 root-specific cis-regulatory elements putatively responsible for OsAER1 root-specific expression. Using the 3082-bp promoter fragment to drive the expression of the GUS reporter transgene confirmed that the OsAER1 promoter is root-specific. Further, the analysis indicated that OsAER1 promoter activity was absent in leaves, petioles and shoots during sprouting, vegetative, booting and generative stages of rice development. In contrast, the promoter activity was present in anthers and aleurone layers of immature seeds 7–20 days after anthesis. Moreover, there was no promoter activity observed in the aleurone layers of mature seeds. The OsAER1 promoter activity is induced by Al-toxicity, NaCl and submergence stresses, indicating the OsAER1 promoter activity is induced by those stresses. Exogenous treatments of transgenic plants carrying the PA-5 promoter construct with abscisic acid and indoleacetic acid also induced expression of the GUS reporter transgene, indicating the role of plant growth regulators in controlling OsAER1 promoter activity. Promoter deletion analysis was conducted to identify the cis-acting elements of the promoter responsible for controlling root-specific expression. The GUS reporter gene was fused with various deletion fragments of the OsAER1 promoter and the resulting constructs were transformed in rice plants to generate transgenic plants. The results of this analysis indicated that cis-acting elements controlling root-specific expression are located between −1562 to −1026bp of the OsAER1 CDS. Here we discusses the results of the conducted analyses, the possible role of OsAER1 in rice growth and development, possible contributions and the potential usage of these findings in future plant research.

Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

<p>Reporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct.</p>

Association of allele variants of IGF-I, GH and PIT-1 genes with production traits of Birkivska Barvista chicken breed

The article considers the questions about association between allelic variants of insulin-like growth factor I (IGF-I), growth hormone (GH) and pituitary transcription factor-1 (PIT-1) genes with the egg productivity of the Borkovskaya Barvistaya chicken breed. Using classical PCR and PCR-RFLP methods, the PstI-polymorphism in the 5'UTR fragment of the IGF-I gene, HinfI-polymorphism in the promoter fragment of IGF-I gene, AluI-polymorphism in the fourth intron of the GH gene and indel polymorphism in the second intron of the PIT-1 gene were determined. As a result of the studies the significant differences in egg weight values on 30 weeks of life for HinfI-polymorphism in the promoter fragment of the IGF-I gene were revealed. For individuals with the C/C genotype was specific the large values of this index compared to A/A (51,7 ± 0,61 vs. 47.9 ± 0,92 respectively). For PstI-polymorphism in the 5'UTR of the IGF-I gene was shown the prevalence in the number of eggs for 12 weeks of productivity for individuals with the C1C1 genotype compared to C2C2 (65,2 ± 3,77 vs. 60,9 ± 1,33), but in this case the differences are not significantly, what can be caused by a small number of individuals with C1C1 genotype (12). There were no significant differences for other loci.

Genetic polymorphism in the promoter of prolactin gene in layer chicken

Genetic polymorphism of promoter (Fragment 1, 2 and 3) of prolactin gene in four strains of White Leghorn chicken layers i.e. IWH, IWI, IWK and layer control was identified. Two alleles (A and B) were found in all the promoter fragments of the gene. In above mentioned layer populations, A allele was predominant. The genotypic and allelic distribution was significantly differed among the populations. Different demographic and divergent parameters were estimated in all the lines. It is concluded that all 3 fragments of promoter of prolactin gene was polymorphic in four strains of White Leghorn layer populations.