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J Stock

Publications and source records attributed to J Stock.

At least 73 records · Page 4Linked to original sources
PubMed1989

Targeted ablation of alpha-crystallin-synthesizing cells produces lens-deficient eyes in transgenic mice.

Genetic ablation techniques were used to study the role of the lens in mammalian eye development. Ablation was accomplished by microinjecting murine eggs with chimeric DNA constructs in which the alpha A-crystallin gene regulatory sequence (-366 to +46) was fused to the highly cytotoxic diphtheria toxin gene coding sequence. For genetic ablation to be successful the promoter regulating expression should be specific and completely silent in cells necessary for normal mouse development. In this report, we describe the generation and analysis of transgenic mice with this readily discernible phenotype: aphakia or eyes without lens. Of the 109 live-born pups, eight carried the transgene and could be grouped according to the apparent severity of eye malformations. Lines 4, 5 and 6 founder (F0) mice had the most severe phenotype. Histological analysis revealed: marked reduction in eye size, total absence of lens, increased retinal cell density and extensive whorling of the retinal fibre layers. The line 1 F0 mouse displayed a distinct lens opacity and lines 2, 3 and 8 F0 mice were mosaics with a relatively mild, but most unusual phenotype. Their eyes contained a small, highly vacuolated lens. The progeny of these mosaics that inherited the transgene, however, again exhibited the severe phenotype. The aberrant structures of the eyes in which complete genetic ablation of the lens has been achieved suggest that the lens plays a pivotal role in the development of multiple components of the murine eye.

Animals↗
PubMed1988

Association of genetic variant of the glucose transporter with non-insulin-dependent diabetes mellitus.

A DNA sequence polymorphism, revealed by digestion of genomic DNA with the endonuclease Xba1 and hybridisation with a complementary DNA clone for a human glucose transporter, yields two alleles (sizes 6.2 kbp, the X1 allele; or 5.9 kbp, the X2 allele). The genotype frequencies were investigated in three non-insulin-dependent diabetic populations. The frequencies (%) of X1.X1, X1.X2, and X2.X2 were 13, 51, and 36 among 89 North European diabetic subjects, and 8, 38, 54 among their 104 controls (chi 2 test p less than 0.02; G-test p less than 0.02). For 53 South European diabetic patients the frequencies were 19, 50, 31, and for their 41 controls they were 2, 58, 40 (chi 2 test p less than 0.02; G-test p less than 0.01). The corresponding figures were 6, 55, 39 for 45 Japanese patients and 0, 28, 72 for a further 49 controls (chi 2 test p less than 0.01; G-test p less than 0.001). The occurrence of the association of the X1 allele with diabetes in three separate populations suggests that the polymorphic site may be close to a diabetogenic locus on chromosome 1.

Aged↗
PubMed1988

Sensory transduction in bacterial chemotaxis involves phosphotransfer between Che proteins.

The CheA protein of the Salmonella typhimurium chemotaxis system is phosphorylated by ATP. Phospho-CheA transfers its phosphoryl group to a second chemotaxis protein, CheY. Unlike phospho-CheA, phospho-CheY is relatively unstable, rapidly decaying to phosphate and CheY. We propose that phosphorylation of CheY may play a role in its function as a tumble regulator to control motor behavior in response to attractant and repellent stimuli.

Adenosine Triphosphate↗
PubMed1988

Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.

The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-related yeast sex factors, and in at least two cases, the alpha-carboxyl group is also methyl esterified. To determine if ras proteins are themselves modified by a similar type of methylation reaction, we incubated rat embryo fibroblasts transformed with p53 and activated Ha-ras oncogenes with L-[methyl-3H]methionine under conditions in which the isotope was converted to the methyl donor S-adenosyl-L-[methyl-3H]methionine. By using an assay that detects methyl ester linkages, we found that immunoprecipitated ras proteins are in fact esterified and that the stability of these esters is consistent with a carboxyl-terminal localization. This methylation reaction may be important in regulating the interaction of ras proteins with plasma membrane components. The presence of analogous carboxyl-terminal tetrapeptide sequences in other proteins may provide a general recognition sequence for lipidation and methylation modification reactions.

Animals↗
PubMed1988

Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism.

We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated glnA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.

Bacterial Proteins↗
PubMed1988

CheA protein, a central regulator of bacterial chemotaxis, belongs to a family of proteins that control gene expression in response to changing environmental conditions.

During bacterial chemotaxis, the binding of stimulatory ligands to chemoreceptors at the cell periphery leads to a response at the flagellar motor. Three proteins appear to be required for receptor-mediated control of swimming behavior, the products of the cheA, cheW, and cheY genes. Here we present the complete nucleotide sequence of the Salmonella typhimurium cheA gene together with the purification and characterization of its protein product. The protein is a 73,000 Mr cytoplasmic constituent. Amino acid-sequence comparisons indicate that it belongs to a family of bacterial regulatory proteins including the products of the cpxA, dctB, envZ, ntrB, phoR, phoM, and virA genes. Each member of this family has a conserved domain of approximately equal to 200 residues within its C terminus. We have previously shown that another chemotaxis protein, CheY, represents a domain of protein structure that has been conserved within a second large family of bacterial regulatory proteins. Each protein of the CheA family seems to function as a regulator of a different CheY homologue. Although each pair of proteins appears to produce a specialized response to a distinct type of stimulus, the relationships in primary structure suggest that a similar molecular mechanism may be involved.

Amino Acid Sequence↗
PubMed1987

N-terminal methylation of proteins: structure, function and specificity.

A common site for the posttranslational modification of proteins is at the N-terminal alpha-amino group. Here we consider the enzymatic addition of one or more methyl groups that has been found to occur in several proteins. Although the methylated proteins have different overall functions, they all appear to be involved in large macromolecular structures such as ribosomes, myofibrils, nucleosomes, pilins, or flagella. Structural features at the N-termini of these methylated proteins suggest that sequences in this region may serve as recognition sites for only a few different types of methylating enzymes. Thus, we propose that three enzymes could account for the N-methylated species so far identified in bacteria, the hypothetical MAK, QP, and pilin methyltransferases, and a single additional enzyme, the hypothetical PK methyltransferase, could account for all of the alpha-amino methylations observed in eukaryotic cells. Finally, we discuss criteria that could be used in conjunction with primary sequence data to predict proteins that might be subject to methylation at their amino termini.

Amino Acid Sequence↗
PubMed1987

A second type of protein methylation reaction in bacterial chemotaxis.

CheZ is the product of one of six genes required for sensory processing in Escherichia coli and Salmonella typhimurium chemotaxis. This 24-kDa cytoplasmic protein is modified by a posttranslational methylation reaction. The modified residue has been identified by analysis of radioactively labeled protein from two-dimensional electrophoretograms and Edman degradation of CheZ protein isolated by immunoaffinity chromatography using anti-CheZ monoclonal antibodies. The methylated group is an N-monomethylmethionine residue at the amino terminus of CheZ. L16, a ribosomal protein that is required for peptidyltransferase activity during protein synthesis, is also methylated at its amino-terminal methionine (Chen, R., Brosius, J., and Wittmann-Liebold, B. (1977) J. Mol. Biol. 111, 173-181). Homologous sequences at the amino termini of L16 and CheZ raise the possibility that a single S-adenosylmethionine-dependent methyltransferase modifies both proteins.

Bacterial Proteins↗
PubMed1987

Identification of a possible nucleotide binding site in CheW, a protein required for sensory transduction in bacterial chemotaxis.

CheW is an essential component of the system which mediates chemotaxis in Salmonella typhimurium and Escherichia coli. Here we report the nucleotide sequence of the cheW gene as well as the purification and characterization of the CheW protein. The DNA sequence predicts a protein of 18,000 molecular weight. The pure protein exhibits an apparent molecular weight of 18,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular sieve chromatography under nondenaturing conditions indicates a molecular weight of approximately 35,000, however. This result suggests that CheW is a homodimer. The predicted amino acid sequence between Thr-128 and Asp-160 fits a consensus exhibited by many proteins which bind purine nucleotides.

Amino Acid Sequence↗
PubMed1987

Active site of the enzyme which demethylates receptors during bacterial chemotaxis.

The CheB methylesterase catalyzes the hydrolysis of glutamyl methyl esters in bacterial chemoreceptor proteins. Studies with residue-specific inhibitors suggest that a cysteine residue is required. The nucleotide sequence of the cheB gene predicts a 349-amino acid protein with cysteine residues at positions 207 and 309. Oligonucleotide-directed mutagenesis was used to change each cysteine to an alanine. Whereas the Cys207-Ala mutation had essentially no effect on esterase activity, the Cys309-Ala mutation caused a complete inactivation of the enzyme. Cys309 is located adjacent to a sequence of amino acids which is characteristic of the beta-alpha-beta motif found in a number of nucleotide binding proteins associated with receptor function in vertebrate tissues. A central feature of this structure is Gly-X-Gly-X-X-Gly. Mutation of the second glycine in this region (Gly284) to a valine also caused a complete loss of esterase activity.

Bacterial Physiological Phenomena↗
PubMed1987

S-adenosylmethionine may not be essential for signal transduction during bacterial chemotaxis.

We previously showed that a mutant strain of Salmonella typhimurium completely deficient in both the chemoreceptor methylating (CheR) and demethylating (CheB) enzymes can still exhibit chemotaxis to aspartate and other attractants (J. Stock, A. Borczuk, F. Chiou, and J. E. B. Burchenal, Proc. Natl. Acad. Sci. USA 82:8364-8368, 1985). We used this cheR cheB mutant to examine the possibility of an additional requirement for S-adenosylmethionine in chemotaxis besides its role in chemoreceptor methylation. A metE mutation was transduced into a cheR cheB double mutant, and the cells were starved for methionine. Despite the fact that intracellular S-adenosylmethionine dropped from approximately 100 microM to less than 0.2 microM, chemotaxis was largely unaffected. In contrast, a corresponding cheR+ cheB+ metE mutant completely lost its chemotaxis ability after being starved for methionine. We conclude from this observation that the primary requirement for S-adenosylmethionine during bacterial chemotaxis is in the methylation of receptor proteins.

Bacterial Proteins↗
PubMed1986

Demethylation of bacterial chemoreceptors is inhibited by attractant stimuli in the complete absence of the regulatory domain of the demethylating enzyme.

The CheB methylesterase catalyzes the demethylation of membrane receptors during chemotaxis in Salmonella typhimurium. The kinetic properties of the full length product of the cheB gene are compared to those of the isolated C-terminal catalytic domain. The fragment has at least a 15-fold higher specific activity than the intact protein. In intact cells receptor demethylation is inhibited by attractants such as L-aspartate. We show here that both forms of the enzyme are similarly inhibited in vitro. Thus, the C-terminal catalytic domain of the CheB protein is sufficient for this aspect of esterase regulation. Inhibition by attractants appears to be caused by changes in receptor conformation rather than by changes in the activity of the demethylating enzyme.

Aspartic Acid↗
PubMed1985

Multiple forms of the CheB methylesterase in bacterial chemosensing.

The methylesterase which catalyzes demethylation of chemotactic membrane receptors in Salmonella typhimurium has been purified and characterized. Two forms of the enzyme have been isolated from cell extracts. One corresponds in molecular weight, Mr = 37,000, and amino acid composition to the predicted product of the structural gene for the methylesterase, cheB. The other is a proteolytic fragment, Mr = 21,000, corresponding to the C-terminal three-fifths of the intact CheB protein. The specific activity of the 21-kDa enzyme is at least 15-fold greater than that of its 37-kDa precursor. We conclude that the CheB protein is composed of at least two structurally distinct portions: a C-terminal catalytic domain, and an N-terminal region which modulates esterase activity.

Amino Acid Sequence↗
PubMed1985

Neither methylating nor demethylating enzymes are required for bacterial chemotaxis.

Clarification of the information processing system in bacterial sensing has been obtained by studying mutants that lack the capacity to modify receptors covalently. The remaining part of the system is able to receive signals from the receptor, to respond with partial adaptation, and to exhibit a chemotactic response. A cycle of chemical reactions analogous to the rhodopsin-transducin cycle in the visual system is shown to provide the proper characteristics to serve as the bridge between receptor and chemotactic output, which allows adaptation in the absence of covalent protein modifications.

Adaptation, Physiological↗
PubMed1985

Homologies between the Salmonella typhimurium CheY protein and proteins involved in the regulation of chemotaxis, membrane protein synthesis, and sporulation.

Chemotactic receptors at the bacterial cell surface communicate with flagellar basal structures to elicit appropriate motor behavior in response to extracellular stimuli. Genetic and physiological studies indicate that the product of the cheY gene interacts directly with components of the flagellar motor to control swimming behavior. We have purified and characterized the Salmonella typhimurium CheY protein and have determined the nucleotide sequence of the cheY gene. Amino acid sequence comparisons showed CheY to be homologous over its entire length (129 residues) to the N-terminal regulatory domain of another protein involved in chemotaxis, the CheB methyl esterase. The entire CheY protein and the regulatory domain of CheB also homologous to the N-terminal portions of the Escherichia coli OmpR and Dye proteins and the Bacillus subtilis Spo0A protein. These homologies suggest an evolutionary and functional relationship between the chemotaxis system and systems that are thought to regulate gene expression in response to changing environmental conditions.

Amino Acid Sequence↗
PubMed1985

Compensatory mutations in receptor function: a reevaluation of the role of methylation in bacterial chemotaxis.

During bacterial chemotaxis membrane receptor proteins are methylated and demethylated at glutamate residues. The generally accepted view is that these reactions play an essential role in the chemosensing mechanism. Strains may be isolated, however, that exhibit chemotaxis in the complete absence of methylation. These are readily obtained by selecting for chemotactic variants of a mutant that completely lacks the methylating enzyme. Methyltransferase activity is not restored; instead, the sensory-motor apparatus is genetically restructured to compensate for the methylation defect. Genetic and biochemical analyses show that the compensatory mutational locus is the structural gene for the demethylating enzyme. Thus, although mutants lacking either the methylating or demethylating enzymes are nonchemotactic, strains defective in both activities exhibit almost-wild-type chemotactic ability.

Bacterial Proteins↗