Genomic structure,expression and evolution of the alfalfa aspartate aminotransferase genes

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.     

The ends and the middle: putting chromosomes together

The three known classes of eukaryotic telomeres share the requirement for an RNA template in their replication. This RNA-templated replication is subject to species-specific differences, such as telomere length and its regulation, which suggest that telomeres may have acquired different additional functions in different organisms. Centromeres show less conservation than do telomeres.     

Sequence of the gene for a NAD(P)-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) from a marine methanotroph Methylobacter marinus A45

Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well.     

Voltage-Gated Potassium Channel Genes Are Clustered in Paralogous Regions of the Mouse Genome

Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, we report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. We find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. We also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes.     

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protien transferase

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella. Correspondence to: R. B. Lingham     

Ultrastructure of Lycopersicon peruvianum leaf explants and streptomycin-resistant shoots on medium containing streptomycin

The effect of streptomycin on morphogenic explants of Lycopersicon peruvianum Mill. was examined microscopically at both the light and ultrastructural level. Early stages in shoot regeneration from leaf explants were distinguished as meristematic tissue at both levels. Small starch grains were observed in the plastids in this tissue but not in plastids in regenerated shoots. In the presence of streptomycin, adventitious shoot regeneration from sensitive leaf strips was inhibited. Large layered bodies were observed within the plastids of sensitive leaf tissue, suggesting the disruption of thylakoid membrane formation. Streptomycin resistant L. peruvianum lines, as well as a chlorophyll-deficient line, were also examined microscopically. The chloroplasts of newly regenerated streptomycin resistant shoots contained well developed internal membranes and conspicuous starch grains. Cells containing a mixture of resistant and sensitive plastids were not observed. The plastids in chlorophyll-deficient tissue completely lacked thylakoid membranes, although small vesicles and intraplastid bodies were seen within the stroma.Abbreviations NMU N-methyl-N-nitrosourea     

Copper deficient rat heart can compensate for doxorubicin-induced oxidant stress

The hypothesis that copper (Cu) alters drug metabolizing enzymes and functions as an antioxidant nutrient in doxorubicin cardiotoxicity was tested. Male Sprague-Dawley rats were fed Cu adequate (⁺Cu; 5 mg Cu/kg of diet), marginally Cu deficient (MCu; 1.2 mg Cu/kg of diet), or severely Cu deficient (⁻Cu; 0.5 mg Cu/kg of diet) diets for 6 wk. Doxorubicin (1, 2, or 4 mg/kg body wt) or saline were administered intraperitoneally 1 time/wk for 4 wk. Compared to control hearts, Cu, Zn superoxide dismutase activity was decreased by 9% in MCu rats and by 21–40% in⁻Cu rats. Glutathione peroxidase activity was elevated 5–15% in⁻Cu rats. Doxorubicin administration increased heart Cu, Zn superoxide dismutase activity in⁺Cu and⁻Cu rats 18 h after the last of 4 injections, but not 18 h after 1 injection. There was no synergism between doxorubicin and Cu deficiency on lipid peroxidation, plasma creatine phosphokinase, cardiac hypertrophy, electrocardiographic abnormalities, or morphological changes. Heart glutathione S-transferase activity was decreased by Cu deficiency, and like Cu, Zn superoxide dismutase activity, returned to normal in⁻Cu rats given doxorubicin. Thus, the Cu deficient rat heart may be able to compensate for doxorubicin-induced oxidant stress by increasing the activity of Cu,Zn superoxide dismutase and glutathione S-transferase.     

Determination of l-α-acetylmethadol, l-α-noracetylmethadol and l-α-dinoracetylmethadol in plasma by gas chromatography—mass spectrometry

A method is described for the simultaneous determination of l-α-acetylmethadol (LAAM) and its N-demethylated metabolites, l-α-noracetylmethadol (norLAAM) and l-α-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography—chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C₁₈), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 μl). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique.