Philip J. Laipis

Mitochondrial DNA copy number in bovine oocytes and somatic cells

Abstract Restriction endonuclease analysis and direct nucleotide sequencing of bovine mitochondrial DNA have revealed a high apparent rate of sequence divergence between maternally related individuals. One possible mechanism that would account for the high rate involves nonuniform amplification and/or segregation of mitochondrial DNA during development of the oocyte. We report here experiments which quantitate the amount of mitochondrial DNA in the bovine oocyte as compared to bovine somatic cells. Total DNA was isolated from purified oocytes, separated by agarose gel electrophoresis, and immobilized on nitrocellulose filters. Hybridization with the complete mitochondrial DNA genome or cloned mitochondrial DNA restriction fragments revealed a 100-fold increase in oocyte mitochondrial DNA as compared to somatic cells. Developing oocytes contained about 4.5 pg or 2.6 × 10 5 copies per cell, whereas primary bovine tissue culture cells contained 0.045 pg or 2.6 × 10 3 copies per cell. These experiments demonstrate directly the amplification of mitochondrial DNA in mammalian oocytes and are consistent with models which could generate mitochondrial DNA polymorphisms by unequal amplification of mitochondrial genomes within an animal.

Analysis of expressed sequence tags from Plasmodium falciparum

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Transcribed heteroplasmic repeated sequences in the porcine mitochondrial DNA D-loop region

The mitochondrial D-loop region of the pig, Sus scrofa, was found to be several hundred base pairs larger than the corresponding region in cow, a related artiodactyl species, primarily because of an insertion containing the tandemly repeated sequence CGTGCGTACA. Porcine mitochondrial DNA from the tissue of a single animal exhibits a large population of length polymorphs, each member of which may have as few as 14 or as many as 29 of these repeat units. This intracellular variability may be due to the repeated and self-complementary properties of this sequence, which would favor mispairing and lead to replication slippage. The repeat domain is unusual in that symmetry properties suggest it may assume alternative conformations including cruciforms and left-handed (Z) DNA. It also appears to be the longest known, naturally occurring, alternating purine-pyrimidine sequence. In order to understand the functional significance of this heteroplasmic domain that potentially disrupts a key regulatory region in the mitochondrial genome, RNA and DNA mapping studies were conducted which located this region between the H-strand replication origin and the putative L-strand transcriptional start site. H-strand RNA analysis demonstrated that this heteroplasmic region is transcribed and, therefore, that priming for H-strand DNA replication in mitochondria is independent of the primer RNA length or secondary structure.

A Murine Model for Human Sepiapterin-Reductase Deficiency

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.

A T7 expression vector optimized for site-directed mutagenesis using oligodeoxyribonucleotide cassettes

Site-directed mutagenesis is widely used to examine structure/function relationships in proteins. We have designed a bacterial expression vector series which is optimized for efficient site-directed mutagenesis and subsequent protein synthesis without intervening subcloning steps. The vectors, derived from the T7 expression vectors of Studier and his collaborators [Studier et al., Methods Enzymol. 185 (1990) 60-89], are small and have a bacteriophage f1 origin of replication for production of single-stranded (ss) DNA. Both single-site mutants [using ssDNA and mutating oligodeoxyribonucleotides (oligos)] and cassette mutants (mutagenesis of a short region by inserting double-stranded oligos into unique restriction sites) are rapidly synthesized and expressed with these vectors. Vector construction and use are detailed with examples showing the expression of the sequences encoding human carbonic anhydrases II and III. Production levels of greater than 60 mg of protein per liter of culture have been obtained.

Structural and kinetic analysis of proton shuttle residues in the active site of human carbonic anhydrase III.

We report the X‐ray crystal structures and rate constants for proton transfer in site‐specific mutants of human carbonic anhydrase III (HCA III) that place a histidine residue in the active‐site cavity: K64H, R67H, and K64H‐R67N HCA III. Prior evidence from the exchange of 18O between CO2 and water measured by mass spectrometry shows each mutant to have enhanced proton transfer in catalysis compared with wild‐type HCA III. However, His64 in K64H and K64H‐R67N HCA III have at most a capacity for proton transfer that is only 13% that of His64 in HCA II. This reduced rate in mutants of HCA III is associated with a constrained side‐chain conformation of His64, which is oriented outward, away from the active‐site zinc in the crystal structures. This conformation appears stabilized by a prominent π stacking interaction of the imidazole ring of His64 with the indole ring of Trp5 in mutants of HCA III. This single orientation of His64 in K64H HCA III predominates also in a double mutant K64H‐R67N HCA III, indicating that the positive charge of Arg67 does not influence the observed conformation of His64 in the crystal structure. Hence, the structures and catalytic activity of these mutants of HCA III containing His64 account only in small part for the lower activity of this isozyme compared with HCA II. His67 in R67H HCA III was also shown to be a proton shuttle residue, having a capacity for proton transfer that was approximately four times that of His64 in K64H HCA III. This is most likely due to its proximity and orientation inward towards the zinc‐bound solvent. These results emphasize the significance of side chain orientation and range of available conformational states as characteristics of an efficient proton shuttle in carbonic anhydrase. Proteins 2007.

Epilepsy in Phenylketonuria: A Complex Dependence on Serum Phenylalanine Levels

Summary:  Purpose: Phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism that frequently results in epilepsy if a low Phe diet was not implemented at birth. The mechanisms by which Phe affects the brain are poorly understood.

PKU is a reversible neurodegenerative process within the nigrostriatum that begins as early as 4 weeks of age in Pahenu2 mice

Phenylketonuria (PKU) is a common genetic disorder in humans that arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination. The neuropathogenesis of the disease has not been completely elucidated, however, oxidative stress is considered to be a key feature of the disease process. Hyperphenylalanemia also adversely affects monoaminergic metabolism in the brain. For this reason we chose to evaluate the nigrostriatum of Pah(enu2) mice, to determine if alterations of monoamine metabolism resulted in morphologic nigrostriatal pathology. Furthermore, we believe that recent developments in adeno-associated virus (AAV)-based vectors have greatly increased the potential for long-term gene therapy and may be a viable alternative to dietary treatment for this metabolic disorder. In this study we identified neurodegenerative changes with regenerative responses in the nigrostriatum of Pah(enu2) mice that are consistent with oxidative injury and occurred as early as 4 weeks of age. These neuropathologic changes were reversed following portal vein delivery of a recombinant adeno-associated virus-mouse phenylalanine hydroxylase-woodchuck hepatitis virus post-transcriptional response element (rAAV-mPAH-WPRE) vector to Pah(enu2) mice and corresponded to rapid reduction of serum Phe levels.

Properties of Intramolecular Proton Transfer in Carbonic Anhydrase III

We investigated the efficiency of glutamic acid 64 and aspartic acid 64 as proton donors to the zinc-bound hydroxide in a series of site-specific mutants of human carbonic anhydrase III (HCA III). Rate constants for this intramolecular proton transfer, a step in the catalyzed dehydration of bicarbonate, were determined from the proton-transfer-dependent rates of release of H2 18O from the enzyme measured by mass spectrometry. The free energy plots representing these rate constants could be fit by the Marcus rate theory, resulting in an intrinsic barrier for the proton transfer of deltaG0++ = 2.2 +/- 0.5 kcal/mol, and a work function or thermodynamic contribution to the free energy of reaction wr = 10.8 +/- 0.1 kcal/mol. These values are very similar in magnitude to the Marcus parameters describing intramolecular proton transfer from His64 and His67 to the zinc-bound hydroxide in mutants of HCA III. That result and the equivalent efficiency of Glu64 and Asp64 as proton donors in the catalysis by CA III demonstrate a lack of specificity in proton transfer from these sites, which is indirect evidence of a number of proton conduction pathways through different structures of intervening water chains. The dominance of the thermodynamic contribution or work function for all of these proton transfers is consistent with the view that formation and breaking of hydrogen bonds in such water chains is a limiting factor for proton translocation.

A physical map of bovine mitochondrial DNA from a single animal.

Mitochondrial DNA has been isolated from the liver of an individual Holstein cow and a physical map has been derived for the 38 cleavage sites made by restriction endonucleases: Ava I, Bam HI, Bgl II, Bst EII, Eco RI, Hha I, Hin dIII, Hpa I, Kpn I, Pst I, Sac I, Sal I, Xba I, and Xho I. Sufficient mitochondrial DNA (approx. 16 mg) could be isolated, allowing this map to serve as the basis for detailed physical, genetic and nucleotide sequence studies in an individual mammal.