Aktivitas Promoter â-aktin Ikan Medaka Jepang (Oryzias latipes) pada Ikan Mas (Cyprinus carpio)

This study was conducted to examine activity of medaka (Oryzias latipes) â-actin promoter (mBP) in common carp(Cyprinus carpio) as the first step towards development of common carp transgenic in country. Gene constructpmBP-hrGFP that consists of mBA promoter and humanized Renilla reniformis green fluorescent protein gene(hrGFP) was injected into cytoplasm of one cell stage of common carp by using microinjector. PmBP-hrGFPconcentration used for microinjection was 50 μg/mL aquabides. Parameters observed were survival rate of embryo(SRe), hatching rate (HR) and expression of hrGFP gene. SRe was calculated before eggs hacthed, while hatchingrate (HR) was after all of eggs hatched. The activity of mBA promoter was analyzed by observation of hrGFP genetransient expression using a fluorescence microscope. The results of experiment showed that SRe (87,5%) andHR (79.2%) of control was respectevily higher than that of injected treatment (75.0% & 61.7%). Expression of hrGFPwas observed firstly at blastula (12 hours after fertilization) to 1-day-old larval stages (24 hours after hatching)with higher gene expression at blastula to late gastrula stages. Percentage of micronjected larvae expressinghrGFP at 6 hours after hatching reached 71.6 ± 6.7%. Conclusion was that mBA promoter could drove hrGFPexpression in common carp, hence it can be used to produce common carp transgenic by changing hrGFP withgenes correlated with important traits in aquaculture.

Developmental Regulation of an Adhesin Gene during Cellular Morphogenesis in the Fungal Pathogen Candida albicans

ABSTRACT Candida albicans expresses specific virulence traits that promote disease establishment and progression. These traits include morphological transitions between yeast and hyphal growth forms that are thought to contribute to dissemination and invasion and cell surface adhesins that promote attachment to the host. Here, we describe the regulation of the adhesin gene ALS3, which is expressed specifically during hyphal development in C. albicans. Using a combination of reporter constructs and regulatory mutants, we show that this regulation is mediated by multiple factors at the transcriptional level. The analysis of ALS3 promoter deletions revealed that this promoter contains two activation regions: one is essential for activation during hyphal development, while the second increases the amplitude of this activation. Further deletion analyses using the Renilla reniformis luciferase reporter delineate the essential activation region between positions −471 and −321 of the promoter. Further 5′ or 3′ deletions block activation. ALS3 transcription is repressed mainly by Nrg1 and Tup1, but Rfg1 contributes to this repression. Efg1, Tec1, and Bcr1 are essential for the transcriptional activation of ALS3, with Tec1 mediating its effects indirectly through Bcr1 rather than through the putative Tec1 sites in the ALS3 promoter. ALS3 transcription is not affected by Cph2, but Cph1 contributes to full ALS3 activation. The data suggest that multiple morphogenetic signaling pathways operate through the promoter of this adhesin gene to mediate its developmental regulation in this major fungal pathogen.

Unmodified Prolactin (PRL) and S179D PRL-Initiated Bioluminescence Resonance Energy Transfer between Homo- and Hetero-Pairs of Long and Short Human PRL Receptors in Living Human Cells

We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP2) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP2 when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb β-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.