Cloning and characterization of the platypus mitochondrial genome

The vertebrate mitochondrial genome is highly conserved in size and gene content. Among the chordates there appears to be one basic gene arrangement, but rearrangements in the mitochondrial gene order of the avian lineages have indicated that the mitochondrial genome may be more variable than once thought. Different gene orders in marsupials and eutherian mammals leave the ancestral mammalian order in some doubt. We have investigated the mitochondrial gene order in the platypus (Ornithorhynchus anatinus), a representative of the third major group of mammals, to determine which mitochondrial gene arrangement is ancestral in mammals. We have found that the platypus mtDNA conforms to the basic chordate gene arrangement, common to fish, amphibians, and eutherian mammals, indicating that this arrangement was the original mammalian arrangement, and that the unusual rearrangements observed in the avians and marsupials are probably lineage-specific. Correspondence to: N.J. Gemmell     

Analysis of the regulation of penicillin biosynthesis in Aspergillus nidulans by targeted disruption of the acvA gene

To analyse the regulation of the biosynthesis of the secondary metabolite penicillin in Aspergillus nidulans, a strain with an inactivated acvA gene produced by targeted disruption was used. acvA encodes -(l--aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), which catalyses the first step in the penicillin biosynthetic pathway. To study the effect of the inactivated acvA gene on the expression of acvA and the second gene, ipnA, which encodes isopenicillin N synthase (IPNS), A. nidulans strain XEPD, with the acvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridization pattern indicating the presence of the disrupted acvA gene, and the fusion genes integrated in single copy at the chromosomal argB locus were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both strains from 24 to 68 h of a fermentation run. In the acvA disrupted strain, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction mechanism of regulation of the penicillin biosynthesis gene ipnA and the third gene aat.     

Phloem translocation of Fe,Cu, Mn,and Zn in Ricinus seedlings in relation to the concentrations of nicotianamine,an endogenous chelator of divalent metal ions,in different seedling parts

During the first 8 days of germination the Ricinus seedling is supplied with all nutrients by the endosperm via phloem transport. In 4- to 8-days-old seedlings the concentrations and contents of Fe, Cu, Mn and Zn, and nicotianamine (NA) in the endosperm, cotyledons, hypocotyl and roots were estimated. From the data obtained translocation rates and flow profiles for the metals were established. The main sink for Fe, Mn and Zn were the cotyledons whereas Cu was mainly imported into the hypocotyl. Maximum flow rates occurred between days 5 and 7, for Zn between days 6 and 8.The time kinetics of NA and divalent metal ion concentrations and contents are interpreted as co-transport. The role of NA as transport vehicle of micronutrients in the sieve tubes is discussed.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.