The influence of ammonium permease activity and carbon source on the uptake of ammonium from simple defined media by Saccharomyces cerevisiae

When growing under defined conditions, cells of the yeast Saccharomyces cerevisiae absorbed ammonium more rapidly with glucose as carbon source than with maltose. Ammonium pool sizes and permease activity were also higher in the glucose-grown cells and the relationship implies that firstly, the sugar is a primary determinant of ammonium permease activity and, secondly, the activity of the permease largely determines both the rate of ammonium uptake and ammonium pool size in the first part of the fermentation.     

The catecholamine binding site of the beta-adrenergic receptor is formed by juxtaposed membrane-spanning domains

The catecholamine binding domain of the turkey erythrocyte beta-adrenergic receptor was mapped by determining the sites of covalent labeling of the purified receptor by two beta-adrenergic photoaffinity reagents, [125I]iodocyanopindolol-diazirine (ICYP-da) and [125I] iodoazidobenzylpindolol (IABP). Both labels were incorporated at two separate sites. By sequencing a labeled peptide, one site of labeling was found to lie at Trp330 in the extracellular half of the seventh membrane span. This position is homologous to the retinal attachment site in rhodopsin. The second labeled site was isolated on an 8000-Da peptide and immunoprecipitated using sequence-directed antibodies. This site lies in membrane spans 3-5. Labeling of the two sites was equal using ICYP-da and 3-10-fold greater in the span 7 site using IABP. These data indicate that the catecholamine binding site is formed from the juxtaposition of span 7 and spans 3-5 in a tertiary structure probably similar to that of rhodopsin.     

Glucose transport throughout fermentation by Saccharomyces cerevisiae NCYC 1108

The rate of fermentation of glucose by a polyploid strain of Saccharomyces cerevisiae growing in a defined salts medium depends on the availability of NH₄+⁺. Its decline after exhaustion of the nitrogen source corresponded with the ability of the cells to accumulate the glucose analogue 2-deoxyglucose. Addition of NH₄+⁺to a nitrogen-depleted culture stimulated both glucose utilization and 2-deoxyglucose uptake. Since stimulation was inhibited by cycloheximide, maintenance of glucose transport during fermentation is dependent on protein synthesis.     

Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry

Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signal-to-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein beta-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.     

Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from Escherichia coli

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.     

MAJOR CLADES OF THE ANGIOSPERMS

Abstract— Our knowledge of fundamental angiosperm interrelationships is still very incomplete. The absence of a narrowly circumscribed gymnosperm outgroup, ideally the sister group, makes character evaluation, necessary for a cladistic analysis, difficult. According to current views the superorder Magnoliiflorae with a number of other groups, for example the monocotyledons, may represent a complex of families near the base of the angiosperms. Interrelationships of groups within the monocotyledons are much better understood than those between groups within the dicotyledons. A cladogram of monocotyledon orders based on earlier work by R. Dahlgren, H. T. Clifford, and F. N. Rasmussen is presented. A data matrix for a sample of the angiosperms with 61 characters for 49 taxa, mostly magnoliifloran and related families, is presented. The characters are polarized mainly according to the current view that the primitive angiosperm morphotype is a woody dicotyledon with strobiloid flowers. As an alternative the matrix is adjusted following W. C. Burger's conjecture that the primitive angiosperm was a herbaceous monocotyledon with trimerous flowers. Both matrices were run in a computerized parsimony analysis, resulting in numerous equally parsimonious solutions. This result is illustrative of the great homoplasy in the available character information, and also of how little actually is known about fundamental angiosperm interrelationships or phylogeny.     

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.      

Discrimination of human placental alkaline phosphatase allelic variants by monoclonal antibodies.

Eighteen monoclonal antibodies were produced by the mouse hybridoma method using purified placental alkaline phosphatase (ALP) as antigen. The ability of the various antibodies to discriminate among allelic variants of the enzyme was tested using a large panel of placental ALPs that had been typed electrophoretically. The panel included sets of samples of each of the six common polymorphic phenotypes as well as a series of rare variants. The reactivity of each antibody with each placental ALP (binding ratio) was determined relative to a single standard placental ALP (type 1) in a quantitative binding assay. The findings for six of the antibodies have already been reported. The results on the other 12 antibodies are presented here, and the combined data on the total series of 18 antibodies are analyzed and discussed. Six of the 18 antibodies showed significantly reduced binding to one or another of the products of the three common alleles. In three cases, the discrimination was reflected by essentially "all-or-none" binding reactions. In the other three cases, the binding differences were less marked but could be demonstrated by quantitative comparisons of the binding ratios. Quantitative binding ratio comparisons also enabled heterozygotes to be differentiated from homozygotes in each case. Some of the antibodies showed reduced binding with certain of the rare variant ALP electrophoretic phenotypes. It is estimated that at a minimum this unselected series of 18 antibodies is directed to at least nine different antigenic determinants on the surface of the placental ALP molecule. The results illustrate the power of monoclonal antibodies to discriminate among allelic variants of enzymes.