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J G Wallis

Publications and source records attributed to J G Wallis.

5 recordsLinked to original sources
PubMed1999

The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids.

Desaturation of fatty acids is an important metabolic process. In mammals, 20-carbon and longer polyunsaturated fatty acids are not only incorporated into cellular membranes in a tissue-specific manner, but also serve as the precursors to synthesis of eicosanoid metabolic regulators. The processes of desaturation and elongation in human liver are well characterized, but an alternate Delta8 desaturation pathway that may be important in certain tissues or in cancer cells is less well examined. The Delta8-desaturase enzyme introduces a double bond at the 8-position in 20-carbon fatty acids that have an existing Delta11 unsaturation. We have isolated the first fatty acid Delta8-desaturase, from the protist Euglena gracilis, in order to explore this alternate pathway. A full-length cDNA was obtained after reverse transcription of mRNA purified from heterotrophically grown Euglena, followed by PCR amplification with primers degenerate to conserved histidine-rich regions of microsomal desaturases. The protein predicted from the cDNA sequence is highly homologous to Delta5 and Delta6 desaturases of Caenhorabditis elegans. When the cDNA was expressed in Saccharomyces cerevisiae, the yeast cultures readily desaturated appropriate 20-carbon fatty acids by inserting an additional double bond at the Delta8-position. The enzyme demonstrated a preference for substrates of metabolic significance, 20:3 Delta11,14,17 and 20:2 Delta11,14. Cloning of a Delta8 fatty acid desaturase offers the opportunity to examine an alternate pathway of long chain fatty acid biosynthesis.

Amino Acid Sequence↗
PubMed1997

Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures.

A synthetic antifreeze protein gene was expressed in plants and reduced electrolyte leakage from the leaves at freezing temperatures. The synthetic AFP was expressed as a fusion to a signal peptide, directing it to the extracytoplasmic space where ice crystallization first occurs. The gene was introduced to Solanum tuberosum L. cv. Russet Burbank by Agrobacterium-mediated transformation. Transformants were identified by PCR screening and expression of the introduced protein was verified by immunoblot. Electrolyte-release analysis of transgenic plant leaves established a correlation between the level of transgenic protein expression and degree of tolerance to freezing. This is the first identification of a phenotype associated with antifreeze protein expression in plant tissue.

Amino Acid Sequence↗
PubMed1995

Genetic evidence of a major role for glucose-6-phosphate dehydrogenase in nitrogen fixation and dark growth of the cyanobacterium Nostoc sp. strain ATCC 29133.

Heterocysts, sites of nitrogen fixation in certain filamentous cyanobacteria, are limited to a heterotrophic metabolism, rather than the photoautotrophic metabolism characteristic of cyanobacterial vegetative cells. The metabolic route of carbon catabolism in the supply of reductant to nitrogenase and for respiratory electron transport in heterocysts is unresolved. The gene (zwf) encoding glucose-6-phosphate dehydrogenase (G6PD), the initial enzyme of the oxidative pentose phosphate pathway, was inactivated in the heterocyst-forming, facultatively heterotrophic cyanobacterium, Nostoc sp. strain ATCC 29133. The zwf mutant strain had less than 5% of the wild-type apparent G6PD activity, while retaining wild-type rates of photoautotrophic growth with NH4+ and of dark O2 uptake, but it failed to grow either under N2-fixing conditions or in the dark with organic carbon sources. A wild-type copy of zwf in trans in the zwf mutant strain restored only 25% of the G6PD specific activity, but the defective N2 fixation and dark growth phenotypes were nearly completely complemented. Transcript analysis established that zwf is in an operon also containing genes encoding two other enzymes of the oxidative pentose phosphate cycle, fructose-1,6-bisphosphatase and transaldolase, as well as a previously undescribed gene (designated opcA) that is cotranscribed with zwf. Inactivation of opcA yielded a growth phenotype identical to that of the zwf mutant, including a 98% decrease, relative to the wild type, in apparent G6PD specific activity. The growth phenotype and lesion of G6PD activity in the opcA mutant were complemented in trans with a wild-type copy of opcA. In addition, placement in trans of a multicopy plasmid containing the wild-type copies of both zwf and opcA in the zwf mutant resulted in an approximately 20-fold stimulation of G6PD activity, relative to the wild type, complete restoration of nitrogenase activity, and a slight stimulation of N2-dependent photoautotrophic growth and fructose-supported dark growth. These results unequivocally establish that G6PD, and most likely the oxidative pentose phosphate pathway, represents the essential catabolic route for providing reductant for nitrogen fixation and respiration in differentiated heterocysts and for dark growth of vegetative cells. Moreover, the opcA gene product is involved by an as yet unknown mechanism in G6PD synthesis or catalytic activity.

Cell Division↗
PubMed1994

Transposon mutagenesis of Nostoc sp. strain ATCC 29133, a filamentous cyanobacterium with multiple cellular differentiation alternatives.

Nostoc sp. strain ATCC 29133 (PCC 73102; Nostoc 29133) is a symbiotically-competent, facultatively heterotrophic, diazotrophic cyanobacterium with the capacity to differentiate specialized cells such as heterocysts, akinetes and hormogonial filaments. We have optimized several methods for physiological and molecular genetic analysis of Nostoc 29133. By use of a Tn5 derivative, Tn5-1063 (Km(r)Bm(r)Sm(r)), delivered by conjugation from Escherichia coli, antibiotic-resistant mutants of Nostoc 29133 were generated at a frequency of approximately 1 x 10(-6), 0.4% of which expressed a nitrogen fixation (heterocyst) defective phenotype. Mutant strain UCD 328 was isolated after co-culture of 86 Nostoc 29133::Tn5-1063 clones with the symbiotic plant partner, Anthoceros punctatus; strain UCD 328 expressed a symbiotic phenotype of increased frequency of hormogonia-dependent infection. The transposon and flanking genomic DNA was recovered from strain UCD 328, the mutation and phenotype reconstructed by homologous recombination in Nostoc 29133, and the transposition site identified from a Nostoc 29133 genomic library. Transposon mutagenesis has thus provided the means for isolation and identification of developmental and symbiotic-specific genes of Nostoc 29133.

Conjugation, Genetic↗