RESUMO
An overarching goal of photosynthesis research is to identify how components of the process can be improved to benefit crop productivity, global food security, and renewable energy storage. Improving carbon fixation has mostly focused on enhancing the CO2 fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This grand challenge has mostly proved ineffective because of catalytic mechanism constraints and required chaperone complementarity that hinder Rubisco biogenesis in alternative hosts. Here we refashion Escherichia coli metabolism by expressing a phosphoribulokinase-neomycin phosphotransferase fusion protein to produce a high-fidelity, high-throughput Rubisco-directed evolution (RDE2) screen that negates false-positive selection. Successive evolution rounds using the plant-like Te-Rubisco from the cyanobacterium Thermosynechococcus elongatus BP1 identified two large subunit and six small subunit mutations that improved carboxylation rate, efficiency, and specificity. Structural analysis revealed the amino acids clustered in an unexplored subunit interface of the holoenzyme. To study its effect on plant growth, the Te-Rubisco was transformed into tobacco by chloroplast transformation. As previously seen for Synechocccus PCC6301 Rubisco, the specialized folding and assembly requirements of Te-Rubisco hinder its heterologous expression in leaf chloroplasts. Our findings suggest that the ongoing efforts to improve crop photosynthesis by integrating components of a cyanobacteria CO2-concentrating mechanism will necessitate co-introduction of the ancillary molecular components required for Rubisco biogenesis.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Nicotiana/genética , Proteínas de Plantas/genética , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/genética , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Clonagem Molecular/métodos , Evolução Molecular Direcionada/métodos , Escherichia coli/metabolismo , Cinética , Engenharia Metabólica/métodos , Modelos Moleculares , Mutação , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Synechococcus/metabolismo , Nicotiana/metabolismo , Transformação GenéticaRESUMO
Heparanase is a ß-d-endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase-deficient (Hpse(-/-) ) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse(-/-) mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse(-/-) mice. Furthermore, the ability of Hpse(-/-) mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Glucuronidase/imunologia , Pneumonia/imunologia , Animais , Western Blotting , Movimento Celular/genética , Células Cultivadas , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica/genética , Expressão Gênica/imunologia , Glucuronidase/deficiência , Glucuronidase/genética , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Pneumonia/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/imunologia , Pele/metabolismoRESUMO
Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago.
Assuntos
Marsupiais/genética , Telômero/genética , Animais , Hibridização In Situ , Cromossomos Sexuais/genética , Homeostase do Telômero/genéticaRESUMO
The Tasmanian devil, a marsupial carnivore, is endangered because of the emergence of a transmissible cancer known as devil facial tumor disease (DFTD). This fatal cancer is clonally derived and is an allograft transmitted between devils by biting. We performed a large-scale genetic analysis of DFTD with microsatellite genotyping, a mitochondrial genome analysis, and deep sequencing of the DFTD transcriptome and microRNAs. These studies confirm that DFTD is a monophyletic clonally transmissible tumor and suggest that the disease is of Schwann cell origin. On the basis of these results, we have generated a diagnostic marker for DFTD and identify a suite of genes relevant to DFTD pathology and transmission. We provide a genomic data set for the Tasmanian devil that is applicable to cancer diagnosis, disease evolution, and conservation biology.