Leo M. Hall

Expression of a foreign gene in Chlamydomonas reinhardtii

Genomic transformation of Chlamydomonas reinhardtii exposed to glass-bead abrasion was accomplished with a chimeric neomycin phosphotransferaseII (NPTII)-encoding gene (nos::npt) flanked by the nopaline synthase promoter and polyadenylation sequences obtained from the Ti plasmid of Agrobacterium tumefaciens. These sequences were in a plasmid (pGA482) which also contained gene nit1 encoding nitrate reductase of C. reinhardtii. Transformants were selected by their ability to grow on medium containing nitrate, and 52% of these was also resistant to kanamycin. Evidence for nos::npt expression includes: (1) hybridization with probes specific for npt, (2) demonstration of NPTII activity after electrophoresis of extracts, and (3) chromatographic identification of the reaction product of NPTII, kanamycin phosphate. The highly biased codon usage in Chlamydomonas does not preclude expression.

Rationalization of some genetic anticodonic assignments

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.

Lactate dehydrogenase isolated from human liver mitochondria: Its purification and partial biochemical characterization

We have demonstrated that lactate dehydrogenase is not solely a cytosolic enzyme by the isolation and purification of the enzyme from the mitochondria of human liver. Treatment of the mitochondria with digitonin reveals the LD activity to be associated with the inner membrane-inner matrix and the outer membrane. The mitochondrial LD consists of two fractions separated by ion exchange and affinity chromatography. The first mitochondrial fraction, LD-Mt1, with isoelectric points of 9.8, 9.6, and 4.8, has subunit components of 14500 and 34000 daltons. The second mitochondrial fraction, LD-Mt2, is similar to cytosolic LD-5 with respect to both isoelectric points and subunit molecular weight. The first mitochondrial fraction, LD-Mt1, has physical characteristics previously associated with the isoenzyme LD-6.

Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride

All earthly creatures use only L-amino acids in template directed protein synthesis. The reason for this exclusive use of the L-isomer is not yet apparent, although recent experiments by Usher and his colleagues have shown some stereoselctivity in the aminoacylation of di- and polynucleotides [1–3]. We have separately reported on intramolecular interactions between hydrophobic amino acid side chains and the adenine ring in aminoacyl adenylates [4]. There was a preferential association of Phe > Leu = Ile > Val with the adenine in these studies, but we made no attempts to address the question of D, L selectivity. Recently, in1H NMR studies of N-acetylphenylalanyl adenylate anhydride, we noticed evidence that both D- and L-isomers of the amino acid were present and, furthermore, that one isomer seemed to be associating with the adenine ring more strongly than the other. Using HPLC, we have separated the two diastereoisomers and have enzymatically determined that the isomer which associates more strongly is the biologically important one, the L-isomer. We present those studies here and discuss the evolutionary significance of this finding.

α-Ketoisocaproate stimulates malate-dependent pyruvate formation in rat heart mitochondria

Abstract In isolated rat heart mitochondria incubated with excess ADP and malate, α-ketoisocaproate (KIC) at 0.05–0.1 mM more than doubled the rate of citrate cycle activity (measured as citrate accumulation in the presence of fluorocitrate). Replacement of fluorocitrate with arsenite increased pyruvate formation stoichiometrically with the previous increase in citrate formation. No enhancement of pyruvate or citrate accumulation was seen when malate was omitted or when KIC was replaced with equimolar leucine. KIC (leucine) did not give rise to acetyl CoA, nor was pyruvate formed by way of oxaloacetate and phosphoenolpyruvate. These findings suggest that KIC regulated the formation of pyruvate from malate by acting as a positive modulator of mitochondrial malic enzyme.