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ItemSpecialized transducing phages for ribosomal protein genes of Escherichia coli(National Academy of Sciences, 1975-01-01) Jaskunas, S. R.; Lindahl, L.; Nomura, M.Specialized lambda transducing phages have been isolated carrying approximately half the ribosomal protein genes of E. coli. These phages carry regions of the bacterial chromosome between aroE and fus. The ribosomal protein genes on these phages have been identified by the stimulation of ribosomal protein synthesis in ultraviolet-irradiated bacteria following infection by the transducing phage, and by the in vitro synthesis of ribosomal proteins in a DNA-dependent protein synthesizing system. The results indicate lambdadspcl probably carries at least 22 ribosomal protein genes and lambdadspc2 at least 26 genes. All these genes are clustered between trkA and strA. At least 13 of them have not been previously mapped. ItemCluster of genes in Escherichia coli for ribosomal proteins, ribosomal RNA, and RNA polymerase subunits(National Academy of Sciences, 1975-07-01) Lindahl, L.; Jaskunas, S. R.; Dennis, P. P.; Nomura, M.This work was supported in part by the College of Agriculture and Life Sciences, University of Wisconsin, and by grants from the National Science Foundation (GB31086) and the National Institute of General Medical Sciences (GM-20427). L.L. is a recipient of a Postdoctoral fellowship from the European Molecular Biology Organization. ItemMapping of ribosomal protein genes by in vitro protein synthesis using DNA fragments of lambda fus3 transducing phage DNA as templates(1977-10-25) Lindahl, Lasse; Post, Leonard; Zengel, Janice; Gilbert, Scott F.; Strycharz, William A.; Nomura, Masayasuhfus3 (or hfus2) transducing phage stimulates the synthesis of 27 ribosomal proteins, elongation factors EF-Tu and EF-G, and RNA polymerase subunit (Y in ultraviolet-irradiated Escherichia roli cells (see a preceding paper, Jaskunas, S. R., Fallon, A. M., and Nomura M. (1977) .I. Bid. Chem. 252, 7323-7336). The presence of the structural genes for these proteins on the hfus3 genome was proven by demonstrating the synthesis of these proteins in a DNA-dependent protein-synthesizing system using Afus3 DNA, or its restriction enzyme fragments, or both, as template. Proteins synthesized in the system were identified by a combination of techniques. including gel electrophoresis, immunochemical reactions, and tryptic peptide analysis. Restriction endonuclease-sensitive sites on kfus3 DNA were mapped, and genes were located using restriction endonuclease fragments of Afus3 DNA, isolated directly or cloned in phage or plasmid vectors, as templates for protein synthesis. Additional information about the locations of the genes was obtained using DNA from insertion and deletion mutants of hfux3. The results obtained in these in oilro experiments (rf. Fig. 2), and those obtained in the in uiuo experiments from preceding papers in this series, are in excellent agreement and map the essential genes on Afun and their promoters as follows (going counter-clockwise on the E. coli chromosome): R,,.. (S12, S7), EF-G, EF-Tu; k,,), SlO, LB, (1~4. L2. L23), (L22. S19), s3, (S17, L16, L29); hUC, L14, L34, L5, S14, S8, L6, LlR, S5, (130, L15); pa, S13, Sll, S4, IY, L17. Re-examination of the results of previous physiological experiments (Dennis, P. P. (1974) J. Mol. Biol. 89, 223- 232) using the present information on r-protein gene organization demonstrates that the increase in the differential synthesis rate of ribosomal proteins during the transition period following the nutritional shift-up is primarily due to the increase in the frequency of initiation of the transcription of ribosomal protein genes. ItemCharacterization of hybrid plasmids carrying individual ribosomal ribonucleic acid transcription units of Escherichia coli(American Society for Microbiology, 1977-12) Kenerley, M. E.; Morgan, E. A.; Post, L.; Lindahl, L.; Nomura, M.We have screened the strains with ColE1 hybrid plasmids constructed by Clarke and Carbon (Cell 9:91-99, 1976) for the presence of ribosomal ribonucleic acid (rRNA) genes on the plasmids and identified 16 strains whose plasmids carry rRNA genes. The structures of these 16 plasmids were compared by heteroduplex analysis, and the plasmids were classified into six groups on the basis of their chromosomal origins. Homology with known transducing-phage deoxyribonucleic acids and genetic mapping have assigned locations on the Escherichia coli chromosome to three of the six groups. These are rrnB near rif at 88 min, rrnC near ilvE at 83 min, and rrnD near aroE at 71 min. A fourth group is probably rrnA at 85 min (T. Ikemura and M. Nomura, Cell, 11:779-793, 1977). We conclude that the minimum number of rRNA transcription units per haploid chromosomes is seven, that is, the six groups identified in this work plus a known operon (rrnE near metA at 89 min) that we failed to find among the hybrid plasmids. This heteroduplex analysis also suggests that there are only two kinds of rRNA operons with respect to their spacer region; three of the six rRNA operon groups studied here have one kind, whereas the remaining three have the other kind. ItemOperon-specific regulation of ribosomal protein synthesis in Escherichia coli(National Academy of Sciences, 1979-12) Lindahl, L.; Zengel, J. M.We have cloned a DNA fragment harboring the genes for ribosomal proteins L2, L4, and L23 on a plasmid vector that contains a lac operator and promoter. The cloned ribosomal protein genes are now under the control of lacOP. Addition of a lac inducer to these cells results in a specific 5- to 10-fold increase in the synthesis of the proteins corresponding to the cloned genes. Within 10 min of this induction, the synthesis of ribosomal proteins S3, S19, L3, L16, L22, and L29 stops almost completely. The genes for all these proteins reside in the same chromosomal operon as L2, L4, and L23. We have seen no dramatic effect on the synthesis of any other ribosomal proteins. Thus, the induction of L2, L4, and L23 results in a specific and rapid decrease in the expression of all (or almost all) genes in their own transcription unit. ItemHigh-efficiency, temperature-sensitive suppression of amber mutations in Escherichia coli(American Society for Microbiology (ASM), 1981-01) Zengel, J. M.; Lindahl, L.We have constructed a high-copy-number plasmid carrying an allele of the supD gene (supD43,74). The plasmid conferred temperature-sensitive suppression of amber mutations. Strains carrying the plasmid exhibited 50 to 60% suppression at 30 degrees C but little or no suppression at 42 degrees C. After a temperature shift from 30 to 42 degrees C the efficiency of suppression decreased gradually over a 60- to 90-min period before reaching the 42 degrees C steady-state level of suppression. ItemFluorescence of crayfish metarhodopsin studied in single rhabdoms(Elsevier Inc., 1981-09) Cronin, T.W.; Goldsmith, T.H.Isolated photoreceptor organelles (rhabdoms) from eyes of crayfish (Procambarus. Orconectes) were examined on a microscope system designed for quantitative measurements of fluorescent. Although fully dark-adapted rhabdoms are nonfluorescent or very weakly fluorescent, an increasing emission appears on exposure to light. Over the 30-fold range of intensities studied, the rate of the appearance of this fluorescence is identical to the rate of formation of metarhodopsin from rhodopsin. Furthermore, the excitation spectra for the observed emission are similar to the absorption spectra of crayfish metarhodopsin at both neutral and acid pH. Finally, the amount of fluorescence observed in rhabdoms previously irradiated with selected wavelengths of light is proportional to the amount of metarhodopsin present in the photosteady state established by the prior irradiation. The emission therefore originates from crayfish metarhodopsin. Fluorescence emission peaks at 670 nm at neutral pH. The quantum efficiency is 1.6 +/- 0.4 X 10(-3). Although emission from other rhodopsin photoproducts has previously been noted, this is the first description of fluorescence from the metarhodopsin chromophore site. ItemOversynthesis of elongation factors G and Tu in Escherichia coli(American Society for Microbiology (ASM), 1982-02) Zengel, J. M.; Lindahl, L.We induced the oversynthesis of elongation factors Tu and G by using multicopy plasmids carrying the structural genes for these proteins under the control of the lac operator-promoter. We found no evidence that accumulation of excess elongation factor Tu or G affects the expression of genes for ribosomal proteins or elongation factors. ItemPhotosensitivity spectrum of crayfish rhodopsin measured using fluorescence of metarhodopsin(Rockefeller University Press, 1982-02-01) Cronin, T. W.; Goldsmith, T. H.Discrepancies exist among spectral measurements of sensitivity of crayfish photoreceptors, their absorption in situ, and the number and absorption spectra of crayfish photopigments that are extracted by digitonin solutions. We have determined the photosensitivity spectrum of crayfish rhodopsin in isolated rhabdoms using long wavelength fluorescence emission from crayfish metarhodopsin as an intrinsic probe. There is no measurable metarhodopsin in the dark-adapted receptor, so changes in the emission level are directly proportional to metarhodopsin concentration. We therefore used changes in metarhodopsin fluorescence to construct relaxation and saturation ("photoequilibrium") spectra, from which the photosensitivity spectrum of crayfish rhodopsin was calculated. This spectrum peaks at or approximately 530 nm and closely resembles the previously measured difference spectrum for total bleaches of dark-adapted rhabdoms. Measurements of the kinetics of changes in rhabdom fluorescence and in transmittance at 580 nm were compared with predictions derived from several model systems containing one or two photopigments. The comparison shows that only a single rhodopsin and its metarhodopsin are present in the main rhabdom of crayfish, and that other explanations must be sought for the multiple pigments seen in digitonin solution. The same analysis shows that there is no detectable formation of isorhodopsin in the rhabdom. ItemRegulation of nitrogen catabolic enzymes in Bacillus spp(American Society for Microbiology, 1982-08) Schreier, Harold J.; Smith, Thomas M.; Bernlohr, Robert W.The levels of the inducible nitrogen catabolic enzymes arginase (L-arginine amidinohydrolase, EC 126.96.36.199) and alanine dehydrogenase (L-alanine:NAD+ oxidoreductase [deaminating], EC 188.8.131.52) from Bacillus licheniformis and histidase (L-histidine ammonia-lyase, EC 184.108.40.206) from Bacillus subtilis and the ammonia assimilatory enzymes from B. licheniformis were determined in cultures grown in the presence of different nitrogen sources. Although the levels of these enzymes were dependent upon the nitrogen source present, induction of the catabolic enzymes in response to the addition of inducer occurred even in the presence of preferred nitrogen sources. Intracellular pool sizes of ammonia, glutamate, glutamine, and alpha-ketoglutarate were measured in continuous cultures of b. licheniformis growing in the presence of different nitrogen sources. A comparison of the pool sizes of these metabolites with the ammonia assimilatory enzyme levels showed that the pools of the metabolites did not change in a manner consistent with their use as regulators of the synthesis of any of these enzymes. ItemControl of diel vertical migration: Photoresponses of a larval crustacean(American Society of Limnology and Oceanography, Inc, 1984-01) Forward, Richard B. Jr.; Cronin, Thomas W.; Stearns, Donald E.The lower threshold for phototaxis of dark‐adapted larvae of the crab Rhithropanopeus harrisii is 10⁻⁷ W·m⁻² at 500‐nm light. This threshold does not change throughout development. In the Newport River estuary (North Carolina), larvae show a pattern of nocturnal vertical migration in which the distribution of later‐stage zoeal larvae centers on the 10⁻⁷ W·m⁻² isolume during the day. The behavioral responses underlying this relationship include a negative geotaxis in darkness which changes to a sinking response in light with a natural underwater distribution. The minimum increase in intensity above the lower threshold which evokes sinking decreases progressively with age. The intensity is well above the phototactic response threshold for stage I zoeae, but within one log unit of threshold for the other three zoeal stages. Thus, during the day larvae are associated with a preferred light level. This association results from a negative geotaxis in darkness that changes to a sinking response upon an increase in light intensity. ItemThe nucleotide sequence of the Escherichia coli fus gene, coding for elongation factor G.(Oxford University Press, 1984-02-24) Zengel, Janice M.; Archer, Richard H.; Lindahl, LasseWe have determined the nucleotide sequence of the Escherichia coli fus gene, which codes for elongation factor G. The protein product of the sequenced gene contains 703 amino acids, with a predicted molecular weight of 77,444. The fus gene shows the nonrandom pattern of codon usage typical of ribosomal proteins and other proteins synthesized at a high level. We have identified several potential promoter sequences within the gene. One of these sequences may correspond to the secondary promoter for expression of the downstream tufA gene (encoding elongation factor Tu) whose activity has been described previously (1,2). A comparison of the nucleotide and amino acid sequences of elongation factors G and Tu reveals a limited but significant homology between the two proteins within the 150 amino acid residues at their amino-terminal ends. ItemRole of attenuation in growth rate-dependent regulation of the S10 r-protein operon of E. coli(The European Molecular Biology Organization, 1984-07) Zengel, J.M.; Archer, R.H.; Freedman, L.P.; Lindahl, L.We have investigated the transcription of the 11 gene S10 ribosomal protein operon of Escherichia coli under various growth conditions. The differential synthesis rate of structural gene message increases 2- to 2.5-fold immediately after a shift-up from glycerol minimal medium to glucose plus amino acids. After the initial increase, the transcription rate goes through several oscillations before reaching the new steady-state rate. By comparing the rates of transcription of leader and structural genes, we conclude that these oscillations are due predominantly to changes in the level of read-through at the S10 attenuator. This regulation of attenuation can account for most of the variations in protein synthesis from the S10 operon after a shift. We also measured the level of read-through in cells growing exponentially in different growth media. Over a 2.5-fold range in growth rates, the read-through changed less than 50%. Thus, regulation of attenuation cannot explain the growth-dependent regulation of ribosomal protein synthesis during steady-state growth. Apparently, additional mechanisms are required to control the expression of the S10 operon in exponentially growing cells. ItemDark regeneration of rhodopsin in crayfish photoreceptors(The Rockefeller University Press, 1984-07-01) Cronin, Thomas W.; Goldsmith, Timothy H .The eyes of crayfish were exposed to lights of known spectral composition, and the course of regeneration was followed in the dark by measuring the content of rhodopsin and metarhodopsin in single rhabdoms isolated at various times after the adaptation, using an assay that is based on the fluorescence of metarhodopsin. Complete recovery requires several days in the dark after intense adaptation to orange light, but requires less than 2 d after blue light exposure. Following an orange light exposure with blue produces recovery kinetics characteristic of the blue light exposure alone. This quickening of recovery occurs whether the receptors are exposed to blue light either immediately or many hours after the original exposure to orange. Conversely, following blue light adaptation with orange leads to slow recovery, which is characteristic of orange alone. Recovery from long-wavelength adaptation is slower principally because many rhabdoms seem to delay the onset of regeneration. We suggest that the regeneration system is itself photosensitive, and after orange light adaptation the supply of active chromophore (presumably 11-cis retinal) limits the rate of recovery. Once started, recovery proceeds slowly and continuously, and the total pigment concentration (rhodopsin plus metarhodopsin) in the rhabdomeric membrane remains approximately constant. Within hours after intense adapting exposures, the rhabdoms become altered in appearance, the surfaces become coated with accessory pigment, and the bands of microvilli are less distinct. These changes persist until recovery of rhodopsin proceeds, which suggests that visual pigment regeneration results from addition of newly synthesized rhodopsin associated with membrane turn-over. ItemUse of the beta-lactamase inhibitor clavulanic acid in the isolation of auxotrophic mutants of Bacillus licheniformis(American Society for Microbiology, 1984-08) P.I., Fields; H.J., Schreier; A.L., Saha; R.W., BernlohrThe isolation of auxotrophic mutants of Bacillus licheniformis, a microbe containing constitutive ,β-lactamase activity, was found to be facilitated by the addition of clavulanic acid and cefotaxime during enrichment. ItemPurification and properties of glutamate synthase from Bacillus licheniformis(American Society for Microbiology, 1984-11) Schreier, Harold; Bernlohr, R. W .Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 220.127.116.11] (GItS) was purified to homogeneity from BaciUus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights, -158,000 and -54,000). The enzyme was found to contain 8.1 ± 1 iron atoms and 8.1 + 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GitS from B. Iicheniformis had a requirement for NADPH, oa-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 ,uM. Nonhyperbolic kinetics were obtained for a-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 ,LM, and the apparent Km values obtained for a-ketoglutarate were 6 and 50 ,uM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DLsulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding 10.5 values of 150, 11, and 250 ,uM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics. ItemRegulation of expression from the glnA promoter of Bacillus subtilis requires the glnA gene product(American Society for Microbiology, 1985-05) Schreier, Harold J.; Fisher, Susan H.; Sonenshein, Abraham L.Expression of the cloned glnA gene [coding for glutamine synthetase (EC 18.104.22.168)] of Bacillus subtilis was 10-fold higher in an Escherichia coli strain grown under nitrogen-limiting conditions than in the same strain under nitrogen-excess conditions. Mutations in the E. coli glnA, glnB, glnD, glnE, glnF, glnG, and glnL genes had no effect on the observed regulation. To test whether sequences within the B. subtilis DNA (3.2 kilobase pairs) were responsible for the observed regulation, a plasmid carrying a transcriptional fusion of the B. subtilis glnA promoter with E. coli lacZ was constructed. beta-Galactosidase levels coded for by this plasmid were found to be negatively regulated in trans by a plasmid carrying the entire B. subtilis glnA gene. Analysis of various deletion plasmids showed that the 1.4-kilobase-pair region encoding glutamine synthetase was necessary for the observed regulation of beta-galactosidase. Plasmids coding for 67% or more of the glutamine synthetase polypeptide gave at least partial repression, but a plasmid carrying 30% of the structural gene, as well as a plasmid carrying a deletion internal to glnA, gave no repression. DNA downstream from glnA (to within 130 base pairs of the end of the gene) was not required for the observed regulation. These results suggest that the glnA gene of B. subtilis is autoregulated, supporting the model for glnA control proposed by Dean et al. [Dean, D. R., Hoch, J. A. & Aronson, A. I. (1977) J. Bacteriol. 131, 981-987]. ItemNucleotide sequence of the alpha ribosomal protein operon of Escherichia coli(Oxford University Press, 1985-06-11) Bedwell, D.; Davis, G.; Gosink, M.; Post, L.; Nomura, M.; Kestler, H.; Zengel, J. M.; Lindahl, L.In Escherichia coli some 19 transcription units encoding the 52 ribosomal proteins are scattered throughout the genome. One of the units, the alpha operon, encodes genes for the ribosomal proteins S13, S11, S4 and L17 as well as the alpha subunit of RNA polymerase. We report here the complete 3.0 kb nucleotide sequence of the alpha operon. In addition, we have determined by S1 nuclease mapping the site of transcription termination in this operon. ItemTranscriptional control of the S10 ribosomal protein operon of Escherichia coli after a shift to higher temperature(American Society for Microbiology, 1985-07) Zengel, J. M.; Lindahl, L.In the 5 to 10 min immediately following a shift from 30 to 42 degrees C, the differential synthesis rates of ribosomal proteins encoded by the 11-gene S10 operon are transiently decreased. This effect results largely from a two- to threefold decrease in the differential rate of transcription of the operon. The inhibition of mRNA synthesis is apparently due to two types of control: (i) initiation of transcription at the S10 promoter is inhibited and (ii) readthrough at the attenuator in the S10 leader is decreased. Both of these effects on transcription are independent of the heat shock regulatory gene, htpR. Furthermore, the inhibition of transcription is observed in both relA+ and relA cells, suggesting that the temperature-induced repression does not require the relA-dependent accumulation of guanosine tetraphosphate (ppGpp). However, recovery from the heat shock was slower in relA+ strains than in relA strains. None of the other ribosomal protein operons that we analyzed showed such a strong decrease in transcription after the heat shock. ItemPrecursor for elongation factor Tu from Escherichia coli(American Society for Microbiology, 1986-02) Lifson, E. R.; Lindahl, L.; Zengel, J. M.The tufA gene, one of two genes in Escherichia coli encoding elongation factor Tu (EF-Tu), was cloned into a ColE1-derived plasmid downstream of the lac promoter-operator. In cells carrying this plasmid, the synthesis of EF-Tu was increased four- to fivefold upon the addition of isopropyl-beta-D-thiogalactopyranoside (an inducer of the lac promoter). This condition led to the synthesis of a novel protein, called pTu, which comigrated with EF-Tu on a sodium dodecyl sulfate-polyacrylamide gel but could be separated on an isoelectric focusing gel, since pTu is slightly more basic than EF-Tu. The synthesis of pTu could also be induced by the synthesis of a hybrid protein containing just the amino-terminal half of the EF-Tu protein. Genetic data suggest that pTu is the product of the tufA and tufB genes. The pTu protein was shown to be related to EF-Tu by gel electrophoresis of tryptic peptides. Pulse-chase experiments suggest that pTu is a precursor of EF-Tu. Interestingly, in a classic membrane fractionation procedure, EF-Tu was found in the cytosolic fraction, whereas pTu was partitioned with the outer membrane.