Vector for pop-in/pop-out gene replacement in Pichia pastoris

Gene replacement in yeast is often accomplished by using a counterselectable marker such as URA3. Although ura3 strains of Pichia pastoris have been generated, these strains are inconvenient to work with because they grow slowly, even in the presence of uracil. To overcome this limitation, we have developed an alternative counterselectable marker that can be used in any P. pastoris strain. This marker is the T-urf13 gene from the mitochondrial genome of male-sterile maize. Previous work showed that expression of a mitochondrially targeted form of T-urf13 in Saccharomyces cerevisiae rendered the cells sensitive to the insecticide methomyl, and similar results have now been obtained with P. pastoris. We have incorporated T-urf13 into a vector that also contains an ARG4 marker for positive selection. The resulting plasmid allows for pop-in/pop-out gene replacement in P. pastoris.     

New evidence for hominid presence in the Lower Pleistocene of the Southern Levant

The site of 'Ubeidiya is located in the Jordan Valley, Israel and has been biochronologically dated to 1.5 m.y.a. It exhibits large lithic and faunal assemblages. Previous published hominid material includes a molar (UB 1701) and I(2) (UB 1700). A recent review of the faunal material from previous excavations has revealed a highly worn hominid right lateral lower incisor (UB 335). The tooth was found in situ in the Lower Pleistocene deposits of stratum I-26a, which is comprised of sand and conglomerates of flint, limestone and basalt indicative of a pebbled lakeshore environment. Taphonomic analysis of the macromammal assemblage indicates high-energy fluvial transport. Paleoecological reconstruction suggests a large woodland fauna with a small percent of open steppe species.UB 335 did not differ significantly from the Lower Pleistocene hominid and modern populations but did differ significantly from all other fossil populations. Two-tailed Student t -test and single classification Model II ANOVA of the buccolingual diameter did not distinguish between Lower Pleistocene species: Homo habilis, H. ergaster and H. cf. erectus. Thus, UB 335 can be identified as a Lower Pleistocene hominid although it cannot be securely assigned to any particular species within that time frame. The current date of the 'Ubeidiya deposits and the location of the site within the Levantine corridor suggests a tenative identification as H. ergaster.     

Role of Pseudomonas putida indoleacetic acid in development of the host plant root system

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.     

Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain

The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.     

Strategies used by rhizobia to lower plant ethylene levels and increase nodulation

Agriculture depends heavily on biologically fixed nitrogen from the symbiotic association between rhizobia and plants. Molecular nitrogen is fixed by differentiated forms of rhizobia in nodules located on plant roots. The phytohormone, ethylene, acts as a negative factor in the nodulation process. Recent discoveries suggest several strategies used by rhizobia to reduce the amount of ethylene synthesized by their legume symbionts, decreasing the negative effect of ethylene on nodulation. At least one strain of rhizobia produces rhizobitoxine, an inhibitor of ethylene synthesis. Active 1-aminocyclopropane-1-carboxylate (ACC) deaminase has been detected in a number of other rhizobial strains. This enzyme catalyzes the cleavage of ACC to alpha-ketobutyrate and ammonia. It has been shown that the inhibitory effect of ethylene on plant root elongation can be reduced by the activity of ACC deaminase.     

Transgenic plants with altered ethylene biosynthesis or perception

The plant hormone ethylene is an essential signaling molecule involved in many plant processes including: germination, flower development, fruit ripening and responses to many environmental stimuli. Moreover, large increases in ethylene levels occur during plant stress responses, fruit ripening and flower wilting. Manipulation of ethylene biosynthesis or perception allows us to modulate these processes and thereby create plants with more robust and/or desirable traits, giving us a glimpse into the role of ethylene in the plant. Here, recent and landmark advances in genetic alteration of members of the ethylene pathway in plants and the physiological consequences of these alterations are examined.     

Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure-function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding "matrix." Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these "free-floating" TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack.     

Molecular basis for expression of common and rare fragile sites

Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.     

Overexpression of secretory phospholipase A(2) causes rapid catabolism and altered tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I

Plasma levels of high density lipoprotein (HDL) cholesterol and its major protein component apolipoprotein (apo) A-I are significantly reduced in both acute and chronic inflammatory conditions, but the basis for this phenomenon is not well understood. We hypothesized that secretory phospholipase A(2) (sPLA(2)), an acute phase protein that has been found in association with HDL, promotes HDL catabolism. A series of HDL metabolic studies were performed in transgenic mice that specifically overexpress human sPLA(2) but have no evidence of local or systemic inflammation. We found that HDL isolated from these mice have a significantly lower phospholipid and cholesteryl ester and significantly greater triglyceride content. The fractional catabolic rate (FCR) of (125)I-HDL was significantly faster in sPLA(2) transgenic mice (4.08 +/- 0.01 pools/day) compared with control wild-type littermates (2.16 +/- 0.48 pools/day). (125)I-HDL isolated from sPLA(2) transgenic mice was catabolized significantly faster than (131)I-HDL isolated from wild-type mice after injection in wild-type mice (p < 0.001). Injection of (125)I-tyramine-cellobiose-HDL demonstrated significantly greater degradation of HDL apolipoproteins in the kidneys of sPLA(2) transgenic mice compared with control mice (p < 0.05). The fractional catabolic rate of [(3)H]cholesteryl ether HDL was significantly faster in sPLA(2)-overexpressing mice (6.48 +/- 0.24 pools/day) compared with controls (4.80 +/- 0.72 pools/day). Uptake of [(3)H] cholesteryl ether into the livers and adrenals of sPLA(2) transgenic mice was significantly enhanced compared with control mice. In summary, these data demonstrate that overexpression of sPLA(2) alone in the absence of inflammation causes profound alterations of HDL metabolism in vivo and are consistent with the hypothesis that sPLA(2) may promote HDL catabolism in acute and chronic inflammatory conditions.