Conformational properties of 2,3-methanopyroglutamic acid in peptides: NMR and x-ray diffraction studies

The effects of replacing L-pyroglutamic acid with the cyclopropane analogue 2,3-methanopyroglutamic acid (2,3-MeGlp) on conformation and enzymatic stability have been investigated in 2,3-MeGlp-NHMe and the novel thyrotropin releasing hormone (TRH) analogue [2,3-MeGlp1]-TRH by x-ray diffraction and nmr. While 2,3-MeGlp-NHMe adopts a folded conformation (small psi angle) in the solid state, several conformations are available to the molecule in solution. 1H-nmr of the diastereomeric mixture [(+/- )-2,3-MeGlp1]-TRH indicates a close orientation of the pyrrolidone and imidazole rings. The 2,3-MeGlp-His amide bond is considerably more stable to pyroglutamate aminopeptidase than the Glp-His bond in TRH.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Colonization by Cladosporium spp. of painted metal surfaces associated with heating and air conditioning systems

Cladosporium cladosporioides and C. hebarum colonized painted metal surfaces of covering panels and register vents of heating, air conditioning and ventilation systems. Hyphae penetrated the paint film and developed characteristic conidiophores and conidia. The colonies were tightly appressed to the metal surface and conidia were not readily detectable via standard air sampling procedures.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun.     

Inactivation of Aspartic Transcarbamylase in Sporulating Bacillus subtilis: Demonstration of a Requirement for Metabolic Energy

The aspartic transcarbamylase (ATCase) activity of Bacillus subtilis cells disappears rapidly from stationary-phase cells prior to sporulation. ATCase activity does not appear in the culture fluid during the stationary phase; hence the enzyme appears to be inactivated in the cells. The enzyme is inactivated normally in two different mutants lacking proteases; the activity is very stable in crude extracts of cells or in the culture fluid. These results suggest that ATCase is not inactivated by the general proteolysis that occurs in sporulating bacteria. The inactivation of ATCase can be completely inhibited after it has begun by oxygen starvation or addition of fluoroacetate. Inhibitors of oxidative phosphorylation and electron transport also interrupt the inactivation of ATCase. The inactivation of ATCase is very slow in two mutant strains that are deficient in enzymes of tricarboxylic acid cycle. Addition of gluconate to stationary cultures of the mutant strains, which is known to restore depleted adenosine 5'-triphosphate pools in these bacteria, also restores inactivation of ATCase. These experiments support the conclusion that the generation of metabolic energy is necessary for the inactivation of ATCase in stationary cells. ATCase activity is stable in growing cells in which ATCase synthesis is repressed by addition of uracil; the enzyme is inactivated normally, however, when such cells cease growing.     

Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.     

Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.     

Site fidelity in predictable and unpredictable habitats

Summary Site fidelity, the tendency to return to a previously occupied location, has been observed in numerous species belonging to at least three phyla. In this paper I develop a general model using dynamic programming to investigate conditions under which fidelity to a previously occupied territory will be advantageous. The results predict that site fidelity should be inversely related to heterogeneity in territory quality and the animal's lifespan and positively related to the cost of changing territories, age and probability of mortality in the habitat. The predictability of reproductive outcome (defined as the probability that next period's outcome will be the same as this period's outcome) also affects site fidelity. In predictable habitats, changing territories may be favoured after a bad previous outcome. In contrast, settlement should be independent of the previous outcome in unpredictable habitats. Individuals should also be site-faithful in unpredictable habitats, as long as the mean territory quality is equal among available territories. I also investigate the success of two potential decision rules (always stay and win-stay: lose-switch) relative to the optimal settlement strategy. The results show that these rules may perform as well as the optimal strategy under certain conditions. The always stay strategy does well in unpredictable habitats, when the mean quality within a territory is equal among territories. In contrast, the win-stay: lose-switch strategy performs best in predictable habitats.