The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.Special issue dedicated to Dr. Santiago Grisolia.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.     

Prochymosin expression in Bacillus subtilis

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.     

Phylogenetic and evolutionary implications of nuclear ribosomal DNA variation in dwarf dandelions (Krigia,Lactuceae, Asteraceae)

Restriction site and length variations of nrDNA were examined for 51 populations of seven species ofKrigia. The nrDNA repeat ranged in size from 8.7 to 9.6 kilobase (kb). The transcribed region, including the two ITSs, was 5.35 kb long in all examinedKrigia populations. In contrast, the size of the nontranscribed IGS varied from 3.35 to 4.25 kb. Eight different types of length-variations were identified among the 51 populations, including distinct nrDNA lengths in the tetraploid and diploid populations of bothK. biflora andK. virginica. However, a few variations were detected among populations of the same species or within a cytotype. All populations ofKrigia sect.Cymbia share a 600 bp insertion in IGS near the 18 S gene, and this feature suggests monophyly of the section. AllKrigia spp. had a conjugated type of subrepeat composed of approximately 75 basepairs (bp) and 125 bp. Base modifications in the gene coding regions were highly conserved among species. Forty-five restriction sites from 15 enzymes were mapped, 24 of which were variable among populations. Only four of the variable sites occurred in the rRNA coding region while 20 variable sites were detected in the noncoding regions. Collectively, 25 enzymes generated about 66 restriction sites in each nrDNA; this amounts to about 4.3% of the nrDNA repeat. A total of 50 restriction sites was variable, 28 of which were phylogenetically informative. Phylogenetic analyses of site mutations indicated that two sections ofKrigia, sect.Cymbia and sect.Krigia, are monophyletic. In addition, relationships among several species were congruent with other sources of data, such as cpDNA restriction site variation and morphology. Both length and restriction site variation supported an allopolyploid origin of the hexaploidK. montana. The average sequence divergence value inKrigia nrDNA was 40 times greater than that of the chloroplast DNA. The rapid evolution of nrDNA sequences was primarily due to changes of the IGS sequences.     

The primary structures of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui

The complete amino acid sequences of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui were determined. The sequences were established by manual sequencing of peptides produced with several proteases as well as by cleavage with dilute HCl. Proteins L16, L23 and L33 consist of 119, 154 and 69 amino acid residues, and their molecular masses are 13538, 16812 and 7620 Da, respectively. The comparison of their sequences with those of ribosomal proteins from other organisms revealed that L23 and L33 are related to eubacterial ribosomal proteins from Escherichia coli and Bacillus stearothermophilus, while protein L16 was found to be homologous to a eukaryotic ribosomal protein from yeast. These results provide information about the special phylogenetic position of archaebacteria.     

Human heart cytochrome c oxidase subunit VIII. Purification and determination of the complete amino acid sequence

Subunit VIII was purified from a preparation of the human heart cytochrome c oxidase and its complete amino acid sequence was determined. The sequence proved to be much more related to that of the bovine liver oxidase subunit VIII than to that found in bovine heart. Our finding of a ‘liver-type’ subunit VIII in the human heart enzyme suggests that either there are no isoforms of human subunit VIII or the human oxidase does not show the type of tissue specificity that has been reported for the oxidase in other mammals.     

Determination of intermediates, products and cleavage site in the reaction between plasminogen activator inhibitor type-2 and urokinases

Several specific inhibitors for plasminogen activators have been isolated from various organs and cell lines, those from human placenta and the human monocyte-like cell line U-937 being virtually identical. The reaction between this type of inhibitor, designated as type-2, and high-M_r and low-M_r urokinase-type plasminogen activators was followed by reversed-phase high-performance liquid chromatography and gel electrophoresis. The components, their stable complexes and their dissociation and cleavage products could be clearly identified in both systems. The amino acid sequence of the inhibitor at the cleavage site was determined to be -Met-Thr-Gly-Arg↓Thr-Gly-His-Gly-. A 35-residue carboxy-terminal fragment was found to be released.     

Restriction endonuclease mapping of ribosomal RNA genes: Sequence divergence and the origin of the tetraploid treefrog Hyla versicolor

Hyla chrysoscelis (2n=24) and H. versicolor (2n=48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the western diploid population and about 50% resembling the eastern population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5 terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species.This research was supported, in part, by NSF Grants CDP-8002341 and PRM-8106947 and by faculty research grants from Miami University to J.C.V.     

Molecular evolution of the carbonic anhydrase genes: Calculation of divergence time for mouse carbonic anhydrase I and II

Summary A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3 untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison.