Mutation Pattern Variation Among Regions of the Primate Genome

We sequenced three argininosuccinate-synthetase-processed pseudogenes (ΨAS-A1, ΨAS-A3, ΨAS-3) and their noncoding flanking sequences in human, orangutan, baboon, and colobus. Our data showed that these pseudogenes were incorporated into the genome of the Old World monkeys after the divergence of the Old World and New World monkey lineages. These pseudogene flanking regions show variable mutation rates and patterns. The variation in the G/C to A/T mutation rate (u) can account for the unequal GC contents at equilibrium: 34.9, 36.9, and 41.7% in the pseudogene ΨAS-A1, ΨAS-A3, and ΨAS-3 flanking regions, respectively. The A/T to G/C mutation rate (v) seems stable and the u/v ratios equal 1.9, 1.7, and 1.4 in the flanking regions of ΨAS-A1, ΨAS-A3, and ΨAS-3, respectively. These ``regional' variations of the mutation rate affect the evolution of the pseudogenes, too. The ratio u/v being greater than 1.0 in each case, the overall mutation rate in the GC-rich pseudogenes is, as expected, higher than in their GC-poor flanking regions. Moreover, a ``sequence effect' has been found. In the three cases examined u and v are higher (at least 20%) in the pseudogene than in its flanking region—i.e., the pseudogene appears as mutation ``hot' spots embedded in ``cold' regions. This observation could be partly linked to the fact that the pseudogene flanking regions are long-standing unconstrained DNA sequences, whereas the pseudogenes were relieved of selection on their coding functions only around 30–40 million years ago. We suspect that relatively more mutable sites maintained unchanged during the evolution of the argininosuccinate gene are able to change in the pseudogenes, such sites being eliminated or rare in the flanking regions which have been void of strong selective constraints over a much longer period. Our results shed light on (1) the multiplicity of factors that tune the spontaneous mutation rate and (2) the impact of the genomic position of a sequence on its evolution. Received: 10 February 1997 / Accepted: 21 April 1997     

Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and β-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as ``TEM-1B like' and ``TEM-2 like' restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the β-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of β-lactam–β-lactamase inhibitor combinations. Received: 18 March 1996 / Accepted: 15 July 1996     

Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora

Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.Abbreviations 2,4-D 2,4-dichloro-phenoxyacetic acid - CaMV Cauliflower mosaic virus - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FDA fluorescein diacetate - HEPES (hydroxyethyl)piperazine(ethanesulfonic acid) - HR hypersensitive reaction - Tris tris (hydroxymethyl)amino-methane     

T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.     

Synthesis and platelet aggregation inhibition activity of a series of enantiomeric bicyclo[3.2.0]heptane-6-oximinoacetic acids (1)

Opening of racemic epoxide (3) with (3S)- or (3R)-dimethyl-3-(dimethyl-t-butylsilyloxy)oct-1-ynyl aluminum gave two regioisomers, which were separated chromatographically. The separated regioisomers, themselves mixtures of chromatographically inseparable diastereoisomers, were converted into their dicobalthexacarbonyl complexes, which were easily resolved and isolated by chromatography. The individual diastereoisomers were deprotected to give bicyclo[3.2.0]heptan-3-ones, whose absolute stereochemistry was assigned using circular dichroism. One of these compounds, (1R,2R,3S,5R,3'S)-3-(3'-hydroxyoct-1'-ynyl)-bicyclo[3.2.0]++ +heptan-2-ol-6- oximinoacetic acid (11a) was 4.5 times more potent than PGE1 in inhibiting the ADP-induced aggregation of human platelets. The next most potent compound in this series was the "ent-15-epi" compound (11b), which was 0.034 times the potency of PGE1 in the platelet aggregation assay.     

Cloning and mapping of Saccharomyces cerevisiae photoreactivation gene PHR1.

The yeast Saccharomyces cerevisiae, like most organisms, is able to directly repair pyrimidine dimers by using a photoreactivating enzyme and visible light. Cells carrying the phr1 mutation were shown previously to be unable to photoreactivate dimers, but neither the map position nor the primary gene product of the PHR1 gene has been determined. We have cloned this gene and determined its map position. A plasmid containing a 6.4-kilobase yeast DNA insert has been isolated and shown to restore photoreactivation in a phr1 strain. A 3.1-kilobase subclone has also been shown to complement phr1. The original plasmid was targeted to integrate into chromosomal DNA at a site homologous to the insert by cutting within the insert. Two of these integrants have been mapped on the right arm of chromosome XV; the integrants have been further mapped at ca. 13 centimorgans from prt1. It has also been independently determined that phr1 maps at this location. Thus, we have determined the map position of PHR1 and also have shown that the plasmid contains PHR1 rather than a suppressor of the phr1 mutation.