709 SummaryEthanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize ( Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-β -1,4-glucanase from Acidothermus cellulolyticus , was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei , also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall.With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T 2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.The "9 ϩ 2" microtubule architecture of the eukaryote axoneme has remained virtually unchanged over millions of years of evolution. Understanding the function of molecules that make up the axoneme is important for elucidating the assembly and activity of these structures that are essential for cell motility. The distinctive arrangement of nine outer doublet microtubules in a circle around a central pair of microtubules is recognizable in electron micrographs of flagella and cilia from plants, algae, protists, and animals. Attached along specific microtubules at precise locations and intervals are ranks of substructures including dynein arms, radial spokes, and central pair projections (25,26). Axonemal dyneins form the inner and outer arm structures that have different functions; the outer arms add power and adjust beat frequency (3,4,10,15,16,24,33); the inner arms generate the axonemal waveform (4,7,14,17,27). To work together efficiently, the multiple dynein isoforms must be locally activated and inactivated at different points in the beat cycle, both around the axoneme and along its length.Structural and genetic evidence implicated the radial spokecentral pair structures as key regulators of dynein activity. The radial spoke heads make transient contact with structures that project from the central pair microtubules (35). The central pair is composed of two microtubules (designated C1 and C2 in algae) and their associated structures which include the central pair projections, central pair bridges linking the two tubules, and central pair caps which are attached to the distal or plus ends of the microtubules.Mutants of the alga Chlamydomon...

No abstract

No abstract

No abstract