Levels Of Transport Activity Research Articles

Role of Serine 263 in the Human Choline Transporter 1 and Congenital Myasthenic Syndrome

Disruptions of the neuromuscular junction (NMJ) result in congenital myasthenic syndromes (CMS). CMS is characterized by a range of phenotypes including muscle weakness, apnoeas, akinesia or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). CHT1 re-uptake of choline is a requirement for acetylcholine synthesis and NMJ function. The 13 transmembrane domain CHT1 protein is also thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive variant c.C788>T in SLC5A7 (NM_021815.4), in a family with a history of CMS using whole exome sequencing. This variant encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether a change in phosphorylation and/or charge/size of position 263 results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetics p.S263D and p.S263E. Upon expression in HEK293 cells and the utilization of cell surface biotinylation experiments, we found that WT, p.S263F and p.S263A mutants were present at the plasma membrane. Radioactive transport assays indicated that the p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non-functional. Immobilized metal affinity electrophoresis suggests that wild-type CHT1 may be more phosphorylated than the p.S263F CHT1 mutant. These results support the essential role of the Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status as well as size/charge at this position.

Phosphorylation of Serine 263 in the Human Choline Transporter 1 alters the transporter’s function

Disruptions of the neuromuscular junction result in congenital myasthenic syndromes (CMS). CMS is characterized by akinesia, apneas or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). The 13 transmembrane domain CHT1 protein is thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive substitution c.C788>T in SLC5A7 in a family with a history of CMS using whole exome sequencing. This mutation encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether 1) the p.S263F substitution alters CHT1 phosphorylation state, and 2) a change in phosphorylation results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetic p.S263D and p.S263E. Upon expression in HEK293 cells, we found that p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non‐functional, suggesting that S263 phosphorylation inhibits choline transport. Immobilized metal affinity electrophoresis supports that wild‐type CHT1 may be more phosphorylated than p.S263F CHT1 mutant. These results support the essential role of Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status.Support or Funding InformationWomen and Children’s Health Research Institute, Stollery Children’s Hospital Foundation, Canadian Institute of Health Research & The University of Alberta

Investigation of compatible solutes synthesis and transport of Virgibacillus halodenitrificans PDB-F2 with complete genome analysis

The salt-tolerant mechanism of compatible solutes in the different microorganisms is always a research hotspot, which can help us understand how organism endures the salty environment. Virgibacillus halodenitrificans PDB-F2 could survive in high salinity and degrade phenol, which is a good candidate for wastewater treatment. This study investigated the salt-tolerant mechanism of compatible solutes of this strain and got an insight into its genetic basis through genome sequencing and analyzing. The results found that Virgibacillus halodenitrificans PDB-F2 endured 12% (w/v) NaCl condition by synthesizing or uptaking ectoine, hydroxyectoine, trehalose, glutamic acid and betaine. Osmoprotective effects of exogenous compatible solutes on this strain were hydroxyectoine > ectoine > L-proline > trehalose > glutamate acid > betaine. Under osmotic shock, the strain had a higher preference for hydroxyectoine than ectoine, and the ectoine transport was stimulated at both levels of transport activity and transcription. The sequencing and analyzing of strain genome showed that this strain contained a circular chromosome (3,869,935 bp) and one plasmid (47,824 bp), revealing the genes related with synthesis and transport of above compatible solutes. This study provided further information on the understanding of salt-tolerant mechanism of Virgibacillus halodenitrificans PDB-F2 by compatible solutes.

Price and income elasticities of demand for passenger transport fuels in Spain. Implications for public policies

The significant increase in passenger transport activity (cars) experienced by Spain and its associated increase in energy consumption have several associated negative aspects, including a greater dependence on foreign energy sources and higher GHG emissions. Therefore, reducing the level of transport activity would bring important socioeconomic and environmental benefits. The aim of this paper, which focuses on energy consumption in the passenger transport, is fourfold: (1) to provide a diagnostic of energy consumption in the Spanish passenger transport system and the related problems; (2) to develop a model to calculate price and income elasticities of demand for transport fuel; (3) to apply this model to the Spanish passenger transport sector; (4) to infer policy recommendations derived from the results of the diagnostic and the model. It is claimed that, in view of those low price elasticities and high income elasticities and if a reduction in the scale of transport activity is deemed socially desirable, a combination of instruments is necessary. Fuel taxes play an important role within this combination. Apart from their long-term effects, the low price elasticity of demand for transport fuel would allow the collection of a significant amount of revenues, which could eventually be earmarked to encourage reductions in private transport demand and modal shifts with other instruments.

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na(+)-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na(+) and/or H(+) electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C-, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11-13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX(3)NEFVA(Y/M/F).

The high-affinity E. coli methionine ABC transporter: structure and allosteric regulation.

The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

Control of the Phosphorylation State of the HPr Protein of the Phosphotransferase System in Bacillus subtilis: Implication of the Protein Phosphatase PrpC

In the Gram-positive bacterium Bacillus subtilis as well as in other firmicutes, the HPr protein of the phosphotransferase system (PTS) has two distinct phosphorylation sites, His-15 and Ser-46. These sites are phosphorylated by the Enzyme I of the PTS and by the ATP-dependent HPr kinase/phosphorylase, respectively. As a result, the phosphorylation state of HPr reflects the nutrient supply of the cell and is in turn involved in several responses at the levels of transport activity and expression of catabolic genes. Most important, HPr(Ser-P) serves as a cofactor for the pleiotropic transcription regulator CcpA. In addition to the proteins that phosphorylate HPr, those that are involved in the dephosphorylation are important in controlling the overall HPr phosphorylation state and the resulting regulatory and physiological outputs. In this study, we found that in addition to the phosphorylase activity of the HPr kinase/phosphorylase, the serine/threonine protein phosphatase PrpC uses HPr(Ser-P) as a target.

Chill Activation of Compatible Solute Transporters in Corynebacterium glutamicum at the Level of Transport Activity

The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K(+) and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10 degrees C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K(+) in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K(+) concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K(+), was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K(+)-mediated process.

Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

We conducted a genome-wide screen in the budding yeast Saccharomyces cerevisiae of 4,792 homozygous diploid deletions to identify genes that function in iron metabolism. Strains unable to grow on iron-restricted medium contained deletions of genes that encode the structural components of the high affinity iron transport system (FET3, FTR1), the iron-sensing transcription factor AFT1 or genes required for the assembly of the transport system. We also identified genes that were not previously known to play a role in iron metabolism. Deletion of the gene CWH36 resulted in a severe growth defect on iron-limited medium, as well as increased sensitivity to Congo red and calcofluor white. Iron transport studies demonstrated that Deltacwh36 cells have an inability to copper load apoFet3p. Furthermore, Deltacwh36 cells demonstrated additional phenotypes including distorted vacuole morphology and altered kinetics of FM4-64 trafficking. We show that Deltacwh36 cells have a defect in vacuolar acidification through the use of the pH-sensitive dye LysoSensor Green DND-189. In Deltacwh36 cells, the vacuolar H+-ATPase is not assembled and there are reduced levels of at least one subunit of the V0 complex. The open reading frame responsible for the Deltacwh36 phenotypes is YCL005W-A. This gene contains two introns, has homologues in other Saccharomyces strains, and shows weak homology to a component of the vacuolar H+-ATPase found in organisms as diverse as insect and cow.

Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities.

Intestinal absorption of folates has been characterized as a facilitative process with a low pH optimum. Studies with intestinal epithelial cells have suggested that this activity is mediated by the reduced folate carrier (RFC1). In this paper, we report on folate transport characteristics in an immortalized rat IEC-6 cell line that was found to exhibit the predominant influx activity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs and high folic acid uptake only at the low pH. For the two clones with the highest level of transport activity, relative MTX influx at the two pHs was reversed. Moreover, the low pH MTX influx activity ([MTX](e) = 0.5 microM) was markedly inhibited by 20 microM folic acid while influx at neutral pH was not. Furthermore, in the presence and absence of glucose at low pH, MTX and folic acid influx activity was inhibited by azide, while MTX influx at pH 7.4 was stimulated by azide in the absence of glucose but was unchanged in the presence of glucose and azide. This was contrasted with the results of transfection of the same RFC1 construct into an L1210 murine leukemia cell line bearing a nonfunctional endogenous carrier. In this case, the activity expressed was only at pH 7.4. These data indicate that RFC1 can exhibit two distinct types of folate transport activities in intestinal cells that must depend on tissue-specific modulators.

Localization and Dynamic Regulation of Biogenic Amine Transporters in the Mammalian Central Nervous System

The monoamines, serotonin, dopamine, norepinephrine, epinephrine and histamine, play a critical role in the function of the hypothalamic–pituitary–adrenal axis and in the integration of information in sensory, limbic, and motor systems. The primary mechanism for termination of monoaminergic neurotransmission is through reuptake of released neurotransmitter by Na+,CI-dependent plasma membrane transporters. A second family of transporters packages monoamines into synaptic and secretory vesicles by exchange of protons. Identification of those cells which express these two families of neurotransmitter transporters is an initial step in understanding what adaptive strategies cells expressing monoamine transporters use to establish the appropriate level of transport activity and thus attain the appropriate efficiency of monoamine storage and clearance. The most recent advances in this field have yielded several surprises about their function, cellular and subcellular localization, and regulation, suggesting that these molecules are not static and most likely are the most important determinants of extracellular levels of monoamines. Here, information on the localization of mRNAs for these transporters in rodent and human brain is summarized along with immunohistochemical information at the light and electron microscopic levels. Regulation of transporters at the mRNA level by manipulation in rodents and differences in transporter site densities by tomographic techniques as an index of regulation in human disease and addictive states are also reviewed. These studies have highlighted the presence of monoamine neurotransmitter transporters in neurons but not in gliain situ.The norepinephrine transporter is present in all cells which are both tyrosine hydroxylase (TH)- and dopamine β-hydroxylase-positive but not in those cells which are TH- and phenyl-N-methyltransferase-positive, suggesting that epinephrine cells may have their own, unique transporter. In most dopaminergic cells, dopamine transporter mRNA completely overlaps with TH mRNA-positive neurons. However, there are areas in which there is a lack of one to one correspondence. The serotonin transporter (5-HTT) mRNA is found in all raphe nuclei and in the hypothalamic dorsomedial nucleus where the 5-HTT mRNA is dramatically reduced following immobilization stress. The vesicular monoamine transporter 2 (VMAT2) is present in all monoaminergic neurons including epinephrine- and histamine-synthesizing cells. Immunohistochemistry demonstrates that the plasma membrane transporters are present along axons, soma, and dendrites. Subcellular localization of DAT by electron microscopy suggests that these transporters are not at the synaptic density but are confined to perisynaptic areas, implying that dopamine diffuses away from the synapse and that contribution of diffusion to dopamine signalling may vary between brain regions. Interestingly, the presence of VMAT2 in vesicles underlying dendrites, axons, and soma suggests that monoamines may be released at these cellular domains. An understanding of the regulation of transporter function may have important therapeutic consequences for neuroendocrine function in stress and psychiatric disorders.

An immortal cell line to study the role of endogenous CFTR in electrolyte absorption.

The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of "endogenous" CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl- conductance (GCl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10(-5) M) significantly increased the Cl- diffusion potential (Vt) generated by a luminally directed Cl- gradient from -15.3 +/- 0.7 mV to -23.9 +/- 1.1 mV, n = 39; and decreased the transepithelial resistance (Rt) from 814.8 +/- 56.3 omega.cm2 to 750.5 +/- 47.5 omega.cm2, n = 39, (n = number of cultures). cAMP activation, anion selectivity (Cl- > I- > gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10(-5) M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 +/- 3.1% and 77.9 +/- 2.6%, respectively, and an increase in Rt by 7.2 +/- 0.8%, n = 36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes.

We have examined the subcellular distribution of three members of the human glucose transporter family expressed in oocytes from Xenopus laevis. Following injection of in vitro-transcribed mRNA encoding the transporter isoform to be studied, we have determined the subcellular localization of the expressed protein by immunofluorescence and by subcellular fractionation coupled with immunoblotting using specific anti-peptide antibodies. We have shown that both the liver-type (GLUT 2) and brain-type (GLUT 3) glucose transporters are expressed predominantly in the plasma membranes of oocytes, and in both cases high levels of glucose transport activity are exhibited. In contrast, the insulin-regulatable glucose transporter (GLUT 4) is localized predominantly to an intracellular membrane pool, and the levels of transport activity recorded in oocytes expressing GLUT 4 are correspondingly lower. The localization of the different transporter isoforms to distinct subcellular fractions mirrors the situation observed in their native cell type and thus demonstrates that oocytes may prove to be a useful system with which to study the targeting signals for this important class of membrane proteins. In addition, the determination of the amounts of the transporters expressed per oocyte together with a knowledge of their Km values has allowed us to estimate the turnover numbers of these transporters. Insulin was without effect on glucose transport in oocytes expressing any of these transporter isoforms. Microinjection of guanosine 5'-[gamma-thio]triphosphate into oocytes expressing GLUT 4 was also without effect on the transport rate.

Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli.

The Escherichia coli uhp T gene encodes an active transport system for sugar phosphates. When the uhp T gene was carried on a multicopy plasmid, amplified levels of transport activity occurred, and growth of these strains was inhibited upon the addition of various sugar phosphates. Two different mechanisms for this growth inhibition were distinguished. Exposure to glucose-6-phosphate, fructose-6-phosphate or mannose-6-phosphate, which enter directly into the glycolytic pathway, resulted in cessation of growth and substantial loss of viability. Cell killing was correlated with the production of the toxic metabolite, methylglyoxal. In contrast, addition of 2-deoxyglucose-6-phosphate, galactose-6-phosphate, glucosamine-6-phosphate or arabinose-5-phosphate, which do not directly enter the glycolytic pathway, resulted in growth inhibition without engendering methylglyoxal production or cell death. Inhibition of growth could result from excessive accumulation of organophosphates in the cell or depletion of inorganic phosphate pools as a result of the sugar-P/Pi exchange process catalysed by UhpT. The phosphate-dependent uptake of glycerol-3-phosphate by the GlpT antiporter was strongly inhibited under conditions of elevated sugar-phosphate transport. There are thus two separate toxic effects of elevated sugar-phosphate transport, one of which was lethal and related to increased flux through glycolysis. It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates.

Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli.

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. We observed a 10-fold increase in the level of transport activity in assays with membrane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced. In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure. The addition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes. We determined that hydrolysis of ATP was directly coupled to maltose uptake. In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated. However, in the remaining experiments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported. Possible explanations for a variable stoichiometry are discussed. These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.

Mutant of Escherichia coli K-12 with defective phosphorylation of two periplasmic transport proteins.

Bacterial periplasmic transport systems require the function of a specific substrate-binding protein, located in the periplasm, and several cytoplasmic membrane transport components. In Escherichia coli K-12, the arginine-ornithine transport system requires an arginine-ornithine-binding protein and the lysine-arginine-ornithine (LAO) transport system includes a LAO-binding protein. Both periplasmic proteins can be phosphorylated by a single kinase. The enzyme exhibits a kinase activity and an ATPase activity. A mutant, defective in the phosphorylation of the arginine-ornithine and the LAO periplasmic proteins, was isolated and characterized. The defective enzymatic activity was reflected in substantially reduced levels of transport activity of the periplasmic transport systems that include each of the binding proteins. The binding proteins, extracted from the mutant, showed no detectable alterations in terms of quantity, electrophoretic mobility, or affinity constants. An apparent Km value of 1.0 mM was calculated for the ATPase activity of the defective enzyme. The ATPase activity of the wild-type enzyme yielded an apparent Km value of 50 microM. The amount of inorganic phosphate incorporated in vivo and in vitro into the binding proteins by the activity of the defective kinase was reduced to very low levels. A structural gene for the phosphorylating enzyme was located near the serA marker on the linkage map of E. coli. These results indicate that phosphorylation of the periplasmic transport protein is obligatorily linked to the normal function of the periplasmic transport system.

Regulation of hexose transport in BALB/c 3T3 preadipose cells: effects of glucose concentration and 12-O-tetradecanoylphorbol-13-acetate.

Like many cell types in culture, both undifferentiated and differentiated BALB/c 3T3 preadipose cells respond to glucose deprivation with an increased uptake of 2-deoxy-D-glucose (deoxyglucose) and 3-O-methyl-D-glucose (methylglucose). Glucose readdition to glucose-deprived cultures resulted in a prompt fall in uptake activity; in undifferentiated cells, a half-maximally effective concentration of glucose was approximately 0.5 mM, while 0.1 mM was ineffective. Several hexoses differed in their efficacy of "deactivating" methylglucose transport in glucose-deprived cells; it appeared that a particular hexose must be metabolized beyond the 6-phosphate form to deactivate the transport system. Previous studies have shown that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates hexose transport in undifferentiated and differentiated BALB/c 3T3 cells. In this study, it was found that TPA (and insulin in differentiated cells) prevented the glucose-induced deactivation of transport activity. Glucose-induced deactivation of transport activity was also prevented by cycloheximide or actinomycin D addition concomitantly with glucose. In glucose-starved cells, agents such as TPA and insulin appear to override a cellular control mechanism sensitive to the external concentration of glucose, so that elevated levels of transport activity are maintained under environmental conditions (i.e., a return to physiological glucose concentrations) that normally induce a fall in transport activity.

Regulation of amino acid transport in chicken embryo fibroblasts by purified multiplication-stimulating activity (MSA).

Enhanced amino acid transport is observed when quiescent cultures of chicken embryo fibroblasts are stimulated to proliferate by the addition of purified multiplication-stimulating activity (MSA). This increase in amino acid transport is an early event occurring prior to the onset of DNA synthesis in stimulated cells. Results indicate that the changes in transport activity, as measured by alpha-aminoisobutyric acid (AIB) uptake, are due to stimulation of only the Na+-dependent A transport system. There is little or no change in the activities of transport systems ASC, L, or Ly+ upon exposure to MSA. A kinetic analysis shows this increased activity is due to a change in Vmax while Km remains unaltered. Continuous exposure to the stimulus is required to maintain the increased level of transport activity and the presence of inhibitors of RNA and protein synthesis significantly inhibits the response. Results also indicate that a similar specific increase in the A transport system is initiated when RSV tsNY68 infected cells are shifted to the permissive temperature. It appears that the A system of mediation is emerging as a strategic regulatory site for cell function.

Duplications of histidine transport genes in Salmonella typhimurium and their use for the selection of deletion mutants

We demonstrate that tandem duplications of the histidine transport operon can be selected by requesting elevated levels of transport activity to be present. Several strains were constructed which contain duplications heterozygotic for either hisJ, hisQ, or hisP. The size of one duplication which was analyzed in detail is about 16 genes, with one end close to the promoter site (dhuA) of the histidine transport operon and, therefore, enclosing about 12 more genes counterclockwise to this operon. Duplication-carrying strains could be utilized for the selection of deletion mutations by requiring both copies of the operon to be rendered defective simultaneously and, therefore, unable to transport into the cell an inhibitory histidine analog, alpha-hydrazino imidazole propionic acid. Over 60% (probably as high as 100%) of the alpha-hydrazino imidazole propionic acid-resistant strains arising in the selection are deletion mutants. The principle of our selection method is generally applicable and will be useful in the accumulation of deletions for mapping and fusing of genes and other purposes.