Two different dihydroorotate dehydrogenases in Lactococcus lactis.

The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic.     

Chloroplast DNA restriction site variation in theVernonieae (Asteraceae), an initial appraisal of the relationship of New and Old World taxa and the monophyly ofVernonia

Chloroplast DNA restriction site variation was examined for 35 taxa in theVernonieae and four outgroup tribes, using 17 restriction enzymes mapped for ca. 900 restriction sites per species; 139 mutations were found to be phylogenetically informative. Phylogenetic trees were constructed using Wagner and weighted parsimony, and evaluated by bootstrap and decay analyses. Relationships of Old and New World taxa indicate complex geographical relationships; there was no clear geographic separation by hemisphere. The relationships between Old and New World Vernonias found here support prior morphological analyses. The sister group to all New and most Old World taxa was composed of a small group of Old World species including yellow-flowered, trinervate-leaved species previously postulated to be basal in the tribe. The majority of both New and Old World taxa are derived from a lineage beginning with the monotypic genusStokesia, an endemic of the southeastern United States. The genusVernonia was also found to be paraphyletic within both the New and Old World. Available data do not support either the separation ofVernonia or the tribeVernonieae into geographically distinct lineages. The pattern of relationships within theVernonieae for taxa from North America, Asia, Africa, Central and South America is most similar to that of several other groups of both plants and animals with a boreotropical origin, rather than an origin in Gondwanaland. Such a pattern of distribution suggests more ancient vicariant events than are routinely postulated for theAsteraceae.     

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

CHLOROPLAST DNA EVIDENCE FOR THE INTERRELATIONSHIPS OF TOMATOES,POTATOES, AND PEPINOS (SOLANACEAE)

We used chloroplast DNA restriction site analysis to test hypotheses of relationships of Solarium subgenus Potatoe (including potatoes and pepinos), two other Solanum, Cyphomandra (the tree tomatoes), and Lycopersicon (the tomatoes). Capsicum and Datura were used as outgroups. The results support two main clades among the taxa we studied: 1) Solanum subgenus Potatoe and Lycopersicon; and 2) other Solanum and Cyphomandra. Within the first clade, the following groups were supported: a) sect. Basarthrum and sect. Anarrhichomenum; b) sect. Etuberosum; c) sect. Petota; d) sect. Juglandifolium, including subsect. Lycopersicoides; and e) the genus Lycopersicon. These results, in combination with an analysis of morphological data, advocate the controversial, but previously suggested, treatment of Lycopersicon as congeneric with Solanum in subgenus Potatoe. Thus, the cultivated tomato will be recognized as Solanum lycopersicum L. Solanum chmielewskii and Solanum lycopersicum var. cerasiforme are proposed as new combinations; Solanum neorickii is proposed as a new name for Lycopersicon parviflorum. Our data also suggest that Cyphomandra should be included within Solanum.     

Chloroplast DNA variation and phylogeny of theRanunculaceae

Restriction site mapping of chloroplast DNA from 31 species representing 26 genera of theRanunculaceae was performed using eleven restriction endonucleases. The chloroplast genome varies in length from approximately 152 to 160 kb. Length variants are frequent in theRanunculaceae and range from usually less than 300 bp to rarely 1.5 kb. The inverted repeat is extended into the large single copy (LSC) region by 4–4.5 kb inAnemone, Clematis, Clematopsis, Hepatica, Knowltonia, andPulsatilla. Several inversions are present in the LSC-region of the cpDNA in all these genera and inAdonis. The frequency of restriction site mutations varies within the chloroplast genome in theRanunculaceae between 4 and 32 mutations per kilobase, and is lowest in the inverted repeat and the regions containing the ATPase-genes and the genespsaA, psaB, psbA, rpoB, andrbcL. A total of 547 phylogenetically informative restriction sites was utilized in cladistic analyses of the family using Wagner, Dollo, and weighted parsimony. These three parsimony analyses result in different tree topologies. Four, six, and one equally most parsimonious trees were obtained with Wagner, Dollo, and weighted parsimony, respectively. The amount of support for the monophyletic groups was evaluated using bootstrapping and decay analysis. All three parsimony methods suggest thatHydrastis is the sister group to the remainder of theRanunculaceae, and that theAnemone-Clematis group, which shares several derived cpDNA rearrangements, is monophyletic. Only a few of the traditional groups in theRanunculaceae are supported by cpDNA restriction side data. Only Dollo parsimony provides support for the hypothesis thatThalictroideae andRanunculoideae are monophyletic.     

A new ATP-binding fold in actin, hexokinase and Hsc70

One of a cell biologist's favourite occupations is to discover the proteins that perform newly described functions in the cell. Very often lately, this has resulted in the identification of protein families whose related amino acid sequences reflect similar functions, but can proteins with totally unrelated sequences have similar structures and functions? In this review, Ken Holmes, Chris Sander and Alfonso Valencia describe the structural similarities between three well-known proteins that have no readily detectable primary sequence similarities but for which X-ray crystallography has revealed very similar structures. A comparison of their structures provides insights into their common mechanisms of action and into protein evolution, and has been used to detect related proteins in sequence data bases.