Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.

Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli. One mutation, designated tyrB, lies at about 80 min on the E. coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase. The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids. In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis. The ilvE gene product alone can reverse a phenylalanine requirement. Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate. These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility. In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.     

Purine and pyrimidine metabolism in human T lymphocytes. Regulation of deoxyribonucleotide metabolism

Purine and pyrimidine deoxyribonucleoside metabolism was studied in G1 and S phase human thymocytes and compared with that of the more mature T lymphocytes from peripheral blood. Both thymocyte populations have much higher intracellular deoxyribonucleoside triphosphate (dNTP) pools than peripheral blood T lymphocytes. The smallest dNTP pool in S phase thymocytes is dCTP (5.7 pmol/10(6) cells) and the largest is dTTP (48 pmol/10(6) cells), whereas in G1 thymocytes, dATP and dGTP comprise the smallest pools. While both G1 and S phase thymocytes have active deoxyribonucleoside salvage pathways, only S phase thymocytes have significant ribonucleotide reduction activity. We have studied ribonucleotide reduction and deoxyribonucleoside salvage in S phase thymocytes in the presence of extracellular deoxyribonucleosides. Based on these studies, we propose a model for the interaction of deoxyribonucleoside salvage and ribonucleotide reduction in S phase thymocytes. According to this model, extracellular deoxycytidine at micromolar concentrations is efficiently salvaged by deoxycytidine kinase. However, due to feedback inhibition of deoxycytidine kinase by dCTP, the maximal level of dCTP which can be achieved is limited. The salvage of both deoxyadenosine and deoxyguanosine (up to 10(-4) M) is completely inhibited in the presence of micromolar concentrations of deoxycytidine, whereas the salvage of thymidine is unregulated resulting in large increases in dTTP levels. Moreover, significant amounts of the salvaged deoxycytidine is used for dTTP synthesis resulting in further increase of dTTP pools. The accumulated dTTP inhibits the reduction of UDP and CDP while stimulating GDP reduction and subsequently also ADP reduction. The end result of the proposed model is that S phase thymocytes in the presence of a wide range of extracellular deoxyribonucleoside concentrations synthesize their pyrimidine dNTP by the salvage pathway, whereas purine dNTPs are synthesized primarily by ribonucleotide reduction. Using the proposed model, it is possible to predict the relative intracellular dNTP pools found in fresh S phase thymocytes.     

Active intestinal chloride secretion in human carriers of cystic fibrosis mutations: an evaluation of the hypothesis that heterozygotes have subnormal active intestinal chloride secretion

To explain the very high frequency of cystic fibrosis (CF) mutations in most populations of European descent, it has been proposed that CF heterozygotes have a survival advantage when infected with Vibrio cholerae or Escherichia coli, the toxins of which induce diarrhea by stimulation of active intestinal chloride secretion. Two assumptions underlie this hypothesis: (1) chloride conductance by the CF transmembrane conductance regulator (CFTR) is the rate-limiting step for active intestinal chloride secretion at all levels of expression, from approximately zero in patients with CF to normal levels in people who are not carriers of a mutation; and (2) heterozygotes have smaller amounts of functional intestinal CFTR than do people who are not carriers, and heterozygotes therefore secrete less chloride when exposed to secretagogues. The authors used an intestinal perfusion technique to measure in vivo basal and prostaglandin-stimulated jejunal chloride secretion in normal subjects, CF heterozygotes, and patients with CF. Patients with CF had essentially no active chloride secretion in the basal state, and secretion was not stimulated by a prostaglandin analogue. However, CF heterozygotes secreted chloride at the same rate as did people without a CF mutation. If heterozygotes are assumed to have less-than-normal intestinal CFTR function, these results mean that CFTR expression is not rate limiting for active chloride secretion in heterozygotes. The results do not support the theory that the very high frequency of CF mutations is due to a survival advantage that is conferred on heterozygotes who contract diarrheal illnesses mediated by intestinal hypersecretion of chloride.     

Role of gammadelta T cells in protecting normal airway function

Since their discovery 15 years ago, the role of gammadelta T cells has remained somewhat elusive. Responses of gammadelta T cells have been found in numerous infectious and non-infectious diseases. New evidence points to gammadelta T cells' functioning in the airways to maintain normal airway responsiveness or tone. In the lung, distinct subsets of gammadelta T cell subsets seem to have specific roles, one subset promoting allergic inflammation, the other serving a protective role.     

Regulation of bidirectional melanosome transport by organelle bound MAP kinase

Regulation of intracellular transport plays a role in a number of processes, including mitosis, determination of cell polarity, and neuronal growth. In Xenopus melanophores, transport of melanosomes toward the cell center is triggered by melatonin, whereas their dispersion throughout the cytoplasm is triggered by melanocyte-stimulating hormone (MSH), with both of these processes mediated by cAMP-dependent protein kinase A (PKA) activity [1, 2]. Recently, the ERK (extracellular signal-regulated kinase) pathway has been implicated in regulating organelle transport and signaling downstream of melatonin receptor [3, 4]. Here, we directly demonstrate that melanosome transport is regulated by ERK signaling. Inhibition of ERK signaling by the MEK (MAPK/ERK kinase) inhibitor U0126 blocks bidirectional melanosome transport along microtubules, and stimulation of ERK by constitutively active MEK1/2 stimulates transport. These effects are specific because perturbation of ERK signaling has no effect on the movement of lysosomes, organelles related to melanosomes [5]. Biochemical analysis demonstrates that MEK and ERK are present on melanosomes and transiently activated by melatonin. Furthermore, this activation correlates with an increase in melanosome transport. Finally, direct inhibition of PKA transiently activates ERK, demonstrating that ERK acts downstream of PKA. We propose that signaling of organelle bound ERK is a key pathway that regulates bidirectional, microtubule-based melanosome transport.     

Identification of Horizontal Gene Transfer from Phylogenetic Gene Trees

We suggest a new procedure to search for the genes with horizontal transfer events in their evolutionary history. The search is based on analysis of topology difference between the phylogenetic trees of gene (protein) groups and the corresponding phylogenetic species trees. Numeric values are introduced to measure the discrepancy between the trees. This approach was applied to analyze 40 prokaryotic genomes classified into 132 classes of orthologs. This resulted in a list of the candidate genes for which the hypothesis of horizontal transfer in evolution looks true.     

A transporter of<Emphasis Type="Italic"> Escherichia coli</Emphasis> specific for<Emphasis Type="SmallCaps"> l</Emphasis>- and<Emphasis Type="SmallCaps"> d</Emphasis>-methionine is the prototype for a new family within the ABC superfamily

An ABC-type transporter in Escherichia coli that transports both l- and d-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds. Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Statistical analysis of the exon-intron structure of higher and lower eukaryote genes

Statistics of the exon-intron structure and splicing sites of several diverse eukaryotes was studied. The yeast exon-intron structures have a number of unique features. A yeast gene usually have at most one intron. The branch site is strongly conserved, whereas the polypirimidine tract is short. Long yeast introns tend to have stronger acceptor sites. In other species the branch site is less conserved and often cannot be determined. In non-yeast samples there is an almost universal correlation between lengths of neighboring exons (all samples excluding protists) and correlation between lengths of neighboring introns (human, drosophila, protists). On the average first introns are longer, and anomalously long introns are usually first introns in a gene. There is a universal preference for exons and exon pairs with the (total) length divisible by 3. Introns positioned between codons are preferred, whereas those positioned between the first and second positions in codon are avoided. The choice of A or G at the third position of intron (the donor splice sites generally prefer purines at this position) is correlated with the overall GC-composition of the gene. In all samples dinucleotide AG is avoided in the region preceding the acceptor site.