The smallest known open reading frame encodes the ribosomal protein L41, which in yeast is composed of only 24 amino acids, 17 of which are arginine or lysine. Because of the unique problems that might attend the translation of such a short open reading frame, we have investigated the properties and the translation of the mRNAs encoding L41. In Saccharomyces cerevisiae L41 is encoded by two linked genes, RPL41A and RPL41B. These genes give rise to mRNAs that have short 5 leaders of 18 and 22 nucleotides and rather long 3 leaders of 203 and 210 nucleotides not including their poly(A) tails. The mRNAs are translated exclusively on monosomes, suggesting that ribosomes do not remain attached to the mRNA after termination of translation. Calculations based on the abundance of ribosomes and of L41 mRNA indicate that the entire translation event, from initiation through termination, must occur in ϳ2 s. Termination of translation after only 25 codons does not subject the mRNAs encoding L41 to nonsense-mediated decay. Surprisingly, despite the L41 ribosomal protein being conserved from the archaea through the mammalia, S. cerevisiae can grow relatively normally after deletion of both RPL41A and RPL41B.

The Akt signaling pathway plays a key role in promoting the survival of various types of cells from stress-induced apoptosis, and different members of the Akt family display distinct physiological roles. Previous studies have shown that in response to UV irradiation, Akt2 is sensitized to counteract the induced apoptosis. However, in response to oxidative stress such as hydrogen peroxide, it remains to be elucidated what member of the Akt family would be activated to initiate the signaling cascades leading to resistance of the induced apoptosis. In the present study, we present the first evidence that knockdown of Akt1 enhances cell survival under exposure to 50 μM H(2)O(2). This survival is derived from selective upregulation and activation of Akt2 but not Akt3, which initiates 3 major signaling cascades. First, murine double minute 2 (MDM2) is hyperphosphorylated, which promotes p53 degradation and attenuates its Ser-15 phosphorylation, significantly attenuating Bcl-2 homologous antagonist killer (Bak) upregulation. Second, Akt2 activation inactivates glycogen synthase kinase 3 beta (GSK-3β) to promote stability of myeloid leukemia cell differentiation protein 1 (MCL-1). Finally, Akt2 activation promotes phosphorylation of FOXO3A toward cytosolic export and thus downregulates Bim expression. Overexpression of Bim enhances H(2)O(2)-induced apoptosis. Together, our results demonstrate that among the Akt family members, Akt2 is an essential kinase in counteracting oxidative-stress-induced apoptosis through multiple signaling pathways.

We designed oligonucleotide gene-specific probes to develop a focused array that can be used to discriminate between neural phenotypes, identify biomarkers, and provide an overview of the process of dopaminergic neuron and glial differentiation. We have arrayed approximately 100 genes expressed in dopaminergic neurons, oligodendrocytes, and astrocytes, an additional 200 known cytokines, chemokines, and their respective receptors, as well as markers for pluripotent and progenitor cells. The genespecific 60-mer 3 biased oligonucleotides for these 281 genes were arrayed in a 25 ؋ 12 format based on function. Using human adult brain substantia nigra, human embryonic stem cells (ESCs), and the differentiated progeny of pluripotent cells, we showed that this array was capable of distinguishing dopaminergic neurons, glial cells, and pluripotent cells by their gene expression profiles in a concentration-dependent manner. Using linear correlation coefficients of input RNA with output intensity, we identified a list of genes that can serve as reporting genes for detecting dopaminergic neurons, glial cells, and contaminating ESCs and progenitors. Finally, we monitored NTera2 differentiation toward dopaminergic neurons and have shown the ability of this array to distinguish stages of differentiation and provide important clues to factors regulating differentiation, the degree of contaminating populations, and stage of cell maturity. We suggest that this focused array will serve as a useful complement to other large-scale arrays in routine assessment of cell properties prior to their therapeutic use. STEM CELLS 2006;24:865-875

The protein phosphatase-2A (PP-2A), one of the major phosphatases in eukaryotes, is a heterotrimer, consisting of a scaffold A subunit, a catalytic C subunit and a regulatory B subunit. Previous studies have shown that besides regulating specific PP-2A activity, various B subunits encoded by more than 16 different genes, may have other functions. To explore the possible roles of the regulatory subunits of PP-2A in vertebrate development, we have cloned the PR55/B family regulatory subunits: β and δ, analyzed their tissue specific and developmental expression patterns in Goldfish ( Carassius auratus). Our results revealed that the full-length cDNA for PR55/Bβ consists of 1940 bp with an open reading frame of 1332 nucleotides coding for a deduced protein of 443 amino acids. The full length PR55/Bδ cDNA is 2163 bp containing an open reading frame of 1347 nucleotides encoding a deduced protein of 448 amino acids. The two isoforms of PR55/B display high levels of sequence identity with their counterparts in other species. The PR55/Bβ mRNA and protein are detected in brain and heart. In contrast, the PR55/Bδ is expressed in all 9 tissues examined at both mRNA and protein levels. During development of goldfish, the mRNAs for PR55/Bβ and PR55/Bδ show distinct patterns. At the protein level, PR55/Bδ is expressed at all developmental stages examined, suggesting its important role in regulating goldfish development. Expression of the PR55/Bδ anti-sense RNA leads to significant downregulation of PR55/Bδ proteins and caused severe abnormality in goldfish trunk and eye development. Together, our results suggested that PR55/Bδ plays an important role in governing normal trunk and eye formation during goldfish development.

It is well established that the protein serine/threonine phosphatase 2A (PP2A) plays very important roles in many different cellular processes, including cell proliferation and differentiation, gene expression, neurotransmission, apoptosis, and aging. PP2A consists of three heterogenic subunits: the scaffold subunit A, the catalytic subunit C, and the regulatory subunit B. While both the scaffold and the catalytic subunits contain only two forms, at least four families of the regulatory subunits, B, B', B'', and B''' have been identified. These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A. To study the functions of the regulatory subunits of PP2A in lower vertebrates, we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit B'delta of PP2A from gold fish, Carassius auratus using 3'-RACE and 5'-RACE cloning strategies. Our results revealed that the full-length B'delta cDNA contains 2415 bp and encodes a protein of 555 amino acids. The B'delta protein displays a very high level of sequence identity with the B'delta regulatory subunit from other species of vertebrates. Regarding its expression pattern, RT-PCR revealed that the highest level of mRNA was detected in brain, a less level detected in liver, spermary, ovary, kidney and gill, and the lowest level detected in the fin. During different developmental stages of gold fish, the highest level of mRNA expression was detected at the stages of two-cell, multiple-cell, blastula and gastrula, and a decreased level of B'gamma mRNA was detected in other developmental stages. At the protein level, the highest expression level of B'delta protein was found in spermary, ovary, brain and heart, a less amount found in liver and the lowest level detected in kidney, gill and fin. Developmentally, B'delta protein was strongly expressed at the stages of two-cell, multiple-cell, blastula, gastrula, neurula, and optic vesicle, and then decreased at the stages of brain differentiation and eye pigmentation. These results suggest that B'delta appears to play a very important role during gold fish development and also in adult tissue homeostasis.