Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance

A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S-transferase (GST)–Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl β-d-thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5°C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55°C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5°C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.

Modulation of Life-span by Histone Deacetylase Genes in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete. Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing. We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity. Deletion of the histone deacetylase gene RPD3 extended life-span. Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3. Similarly, the effect of the rpd3Δ on life-span did not depend on an intact Sir silencing complex. In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging. Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3. The hda1Δ sir3Δ resulted in an increase in silencing, but only at the rDNA locus. Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Δ single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant. Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population. The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span. Our results are consistent with the loss of rDNA silencing leading to aging in yeast. The functions of RPD3 and HDA1 do not overlap entirely. RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process.

A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins

The Krüppel-associated box A (KRAB-A) domain is an evolutionarily conserved transcriptional repressor domain present in approximately one-third of zinc finger proteins of the Cys2-His2 type. Using the yeast two-hybrid system, we report the isolation of a cDNA encoding a novel murine protein, KRAB-A interacting protein 1 (KRIP-1) that physically interacts with the KRAB-A region. KRIP-1 is a member of the RBCC subfamily of the RING finger, or Cys3HisCys4, family of zinc binding proteins whose other members are known to play important roles in differentiation, oncogenesis, and signal transduction. The KRIP-1 protein has high homology to TIF1, a putative modulator of ligand-dependent activation function of nuclear receptors. A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult mouse tissues studied. When a GAL4–KRIP-1 fusion protein is expressed in COS cells with a chloramphenicol acetyltransferase reporter construct with five GAL4 binding sites, there is dose-dependent repression of transcription. Thus, KRIP-1 interacts with the KRAB-A region of C2H2 zinc finger proteins and may mediate or modulate KRAB-A transcriptional repressor activity.

Atmospheric energy for subsurface life on Mars?

The location and density of biologically useful energy sources on Mars will limit the biomass, spatial distribution, and organism size of any biota. Subsurface Martian organisms could be supplied with a large energy flux from the oxidation of photochemically produced atmospheric H2 and CO diffusing into the regolith. However, surface abundance measurements of these gases demonstrate that no more than a few percent of this available flux is actually being consumed, suggesting that biological activity driven by atmospheric H2 and CO is limited in the top few hundred meters of the subsurface. This is significant because the available but unused energy is extremely large: for organisms at 30-m depth, it is 2,000 times previous estimates of hydrothermal and chemical weathering energy and far exceeds the energy derivable from other atmospheric gases. This also implies that the apparent scarcity of life on Mars is not attributable to lack of energy. Instead, the availability of liquid water may be a more important factor limiting biological activity because the photochemical energy flux can only penetrate to 100- to 1,000-m depth, where most H2O is probably frozen. Because both atmospheric and Viking lander soil data provide little evidence for biological activity, the detection of short-lived trace gases will probably be a better indicator of any extant Martian life.

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of γ-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Δ6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Δ5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

Rapid induction of senescence in human cervical carcinoma cells

Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage

DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD+, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.

Keeping Mars warm with new super greenhouse gases

Our selection of new super greenhouse gases to fill a putative “window” in a future Martian atmosphere relies on quantum-mechanical calculations. Our study indicates that if Mars could somehow acquire an Earth-like atmospheric composition and surface pressure, then an Earth-like temperature could be sustained by a mixture of five to seven fluorine compounds. Martian mining requirements for replenishing the fluorine could be comparable to current terrestrial extraction.

Circularly permuted green fluorescent proteins engineered to sense Ca2+

To visualize Ca2+-dependent protein–protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named “pericam,” was fluorescent and its spectral properties changed reversibly with the amount of Ca2+, probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, “flash-pericam” became brighter with Ca2+, whereas “inverse-pericam” dimmed. On the other hand, “ratiometric-pericam” had an excitation wavelength changing in a Ca2+-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca2+ dynamics, such as Ca2+ oscillations in the cytosol and the nucleus. Ca2+ imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca2+ ions across the nuclear envelope. Then, free Ca2+ concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca2+ transients caused rapid changes in the concentration of mitochondrial Ca2+. Finally, a “split-pericam” was made by deleting the linker in the flash-pericam. The Ca2+-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.

E2F-dependent transcription of the raf proto-oncogene during Drosophila development

D-raf, a Drosophila homolog of the raf proto-oncogene, has diverse functions throughout development and is transcribed in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation. The expression pattern resembles those of S phase genes, which are regulated by E2F transcription factors. In the 5′-flanking region of D-raf, four sequences (E2F sites 1–4) similar to the E2F recognition sequence were found, one of them (E2F site 3) being recognized efficiently by Drosophila E2F (dE2F) in vitro. Transient luciferase expression assays confirmed activation of the D-raf gene promoter by dE2F/dDP. Expression of Draf–lacZ was greatly reduced in embryos homozygous for the dE2F mutation. These results suggest that dE2F is likely to be an important regulator of D-raf transcription.

Even-odd alternation in mass spectrum of thymine and uracil clusters: Evidence of intracluster photodimerization

Multiphoton ionization of thymine and uracil clusters generated by a supersonic molecular beam gave rise to a remarkable alternation of mass spectral intensities between even- and odd-numbered clusters. Such alternation was observed in clusters of up to 30 molecules. Excitation to the two lowest electronically excited states seemed to be a strong prerequisite. In view of the well known photodimerization reaction of thymine and uracil in the bulk phase, it is proposed that such alternation in the mass spectral intensity resulted from formation of photodimer units within the cluster on intense UV irradiation. Several analogues of thymine with no known propensity for photodimerization in the bulk phase did not exhibit any sign of such alternation in the cluster mass spectrum. The intrinsic UV window for photodimerization, and hence photoinduced mammalian mutagenesis, was estimated to be approximately 210–280 nm, significantly narrower than the previously reported bulk values of 150–300 nm.

Recursive partitioning for tumor classification with gene expression microarray data

Precise classification of tumors is critically important for cancer diagnosis and treatment. It is also a scientifically challenging task. Recently, efforts have been made to use gene expression profiles to improve the precision of classification, with limited success. Using a published data set for purposes of comparison, we introduce a methodology based on classification trees and demonstrate that it is significantly more accurate for discriminating among distinct colon cancer tissues than other statistical approaches used heretofore. In addition, competing classification trees are displayed, which suggest that different genes may coregulate colon cancers.

Eficiência de uso de nitrogênio pela cana-de-açúcar: diferenças genotípicas, preferência por amônio e emissão de N2O

O uso ineficiente do nitrogênio (N) é um problema global, que pode diminuir a sustentabilidade da produção agrícola da cana-de-açúcar para fins energéticos devido às emissões de N2O oriundas da fertilização nitrogenada. A eficiência de utilização de nitrogênio (EUN) tem sido pouco estudada em genótipos de cana-de-açúcar, o que pode ser preocupante devido a importância da cultura para o país. O objetivo desse estudo foi avaliar a EUN de genótipos de cana-de-açúcar, verificando se genótipos contrastantes tem preferência de absorção por amônio ao invés de nitrato, e checar se extratos radiculares de Brachiaria humidicola e Saccharum spontaneum aumentam a eficiência de utilização de N pela cultura. Para isso três experimentos foram desenvolvidos em condições controladas em casa-de-vegetação e câmara de crescimento. No primeiro deles, 18 genótipos foram submetidos a dois níveis de N: limitante (baixo N) e suficiente (alto N). Posteriormente, quatro genótipos selecionados quanto a EUN do primeiro experimento, foram cultivados em câmara de crescimento por 69 dias, onde receberam quantidades conhecidas de N-fertilizante marcado ou no 15NH4+ ou no 15NO3-, sendo avaliados 24 e 72 horas após a aplicação do fertilizante marcado. Um terceiro ensaio foi realizado, em que extratos radiculares de Brachiaria humidicola (BCH) e Saccharum spontaneum (SCS) foram aplicados ao solo com o intuito de verificar seu potencial como inibidor da nitrificação, comparando-os com a dicianodiamida (DCD), e seu efeito para o aumento do aproveitamento do N pela cana-de-açúcar. No experimento de EUN verificou-se grande variação entre os genótipos. Dos 18 genótipos avaliados, seis foram classificados como responsivos a N e eficientes na utilização de N, três foram não eficientes e responsivos; dois foram eficientes e não responsivos, e sete não eficientes e não responsivos. Esse resultado comprova que a recomendação da adubação nitrogenada pode realmente estar pouco precisa, pois não leva em consideração a EUN de cada genótipo, sendo realizada uma aplicação genérica. As medições de fotossíntese mostraram que os genótipos mais eficientes na utilização do N, não necessariamente possuem as taxas fotossintéticas mais elevadas, estando a EUN diretamente relacionada à quantidade de raízes das plantas. No segundo trabalho, verificou-se que o N na planta proveniente do fertilizante (NPPF), 72 horas após a aplicação do N, foi 36% menor quando a fonte marcada foi NO3-, atestando que a cana-de-açúcar tem preferência de absorção por amônio em relação ao nitrato nos primeiros dias após a adubação nitrogenada. Por fim, os resultados do terceiro ensaio mostraram que a aplicação de sulfato de amônio (SA), associado ao inibidor sintético DCD, manteve os valores de N-NO3- baixos ao longo de todo período de avaliação (durante 60 dias), enquanto que com SA, SA+BCH e SA+SCS houve um aumento na concentração de nitrato no solo já a partir de 15 dia após a fertilização. O uso de DCD reduziu o fluxo médio de N2O durante o período de avaliação em relação ao uso isolado do fertilizante, o que não foi observado com o uso de extratos de raízes de Brachiaria humidicola e Saccharum spontaneum

Crypto at the Time of Surveillance: Sharing with the Cloud

These days as we are facing extremely powerful attacks on servers over the Internet (say, by the Advanced Persistent Threat attackers or by Surveillance by powerful adversary), Shamir has claimed that “Cryptography is Ineffective”and some understood it as “Cryptography is Dead!” In this talk I will discuss the implications on cryptographic systems design while facing such strong adversaries. Is crypto dead or we need to design it better, taking into account, mathematical constraints, but also systems vulnerability constraints. Can crypto be effective at all when your computer or your cloud is penetrated? What is lost and what can be saved? These are very basic issues at this point of time, when we are facing potential loss of privacy and security.