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N Brot

Publications and source records attributed to N Brot.

At least 73 records · Page 4Linked to original sources
PubMed1988

Regulation of ribulose bisphosphate carboxylase expression in Rhodospirillum rubrum: characteristics of mRNA synthesized in vivo and in vitro.

The synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in Rhodospirillum rubrum was regulated by the CO2 concentration in the culture medium. The specific activity of RuBPCase in cells grown photolithotrophically in low concentrations of CO2 (1.5%) was five to ten times higher than that in cultures grown at high concentrations of CO2 (10%). Increased enzyme activity was reflected by an increase in both RuBPCase mRNA and RuBPCase protein. RuBPCase expression was also studied in vitro with a plasmid-borne genomic clone (pRR117) as the template in a partially defined Escherichia coli system containing either E. coli or R. rubrum RNA polymerase. With both enzymes there was excellent synthesis of RuBPCase mRNA, but no significant synthesis of RuBPCase was detected. The promoter region of the RuBPCase gene was sequenced, and mRNA start sites were mapped. A single major in vivo transcriptional start site was detected in RuBPCase mRNA extracted from R. rubrum. However, transcripts synthesized from pRR117 in vitro or from E. coli transformed with pRR117 started at upstream sites that were different from the in vivo transcription site. Two major features of the RuBPCase promoter region are three 6-base-pair direct repeats and a 31-base-pair region of dyad symmetry.

Base Sequence↗
PubMed1987

Association between lupus psychosis and anti-ribosomal P protein antibodies.

In 18 of 20 patients with psychosis secondary to systemic lupus erythematosus (SLE), autoantibodies to ribosomal P proteins were detected by immunoblotting and measured with a new radioimmunoassay using a synthetic peptide as antigen. The frequency of anti-P was not increased in patients with other central nervous system manifestations of SLE (3 of 20, by radioimmunoassay), in patients with transient behavioral abnormalities due to SLE (none of 8), in patients with psychosis who did not have SLE (none of 13), or in normal controls (none of 20). In four of five paired serum samples, anti-P-peptide antibody levels increased 5-fold to 30-fold during the active phase of lupus psychosis. Longitudinal studies of anti-P activity in two patients with psychosis revealed that anti-P levels increased before and during the active phases of psychosis but not during sepsis or other exacerbations of SLE, and that the elevations were selective for anti-P antibodies, as opposed to anti-DNA antibodies. Longitudinal studies of anti-P activity in two patients with anti-P but without psychosis showed less than threefold changes in anti-P levels despite exacerbations of disease. We conclude that anti-P is associated with lupus psychosis and that synthetic peptide antigens may be useful for the detection and measurement of autoantibodies to intracellular proteins.

Adolescent↗
PubMed1987

In vitro expression of the Escherichia coli nusA-infB operon.

The expression of the nusA-infB operon has been investigated using an in vitro system based on the formation of the first dipeptide of the gene product. A series of plasmids containing various deletions of the operon were used as templates in this study. Of the four genes coding for protein products, 15Ka, nusA, infB, and 15Kb, only 15Ka was not expressed in this dipeptide system. The initial dipeptides for the other gene products, fMet-Asn (pnusA), fMet-Thr (IF-2 alpha), and fMet-Ala (p15Kb), were synthesized even from plasmids lacking the primary promoters. It appears that secondary (internal) promoters in the operon can efficiently direct the expression of these genes. No regulation of the expression was observed with IF-2 alpha, but pnusA inhibited the expression of the nusA gene (autoregulation) as well as the p15b gene. Experiments using an uncoupled system indicated that the effect of pnusA on nusA expression was at the level of transcription, but that both a transcriptional and a post-transcriptional effect of pnusA was seen on 15Kb expression.

Bacterial Proteins↗
PubMed1987

Lupus anti-ribosomal P peptide antibodies show limited heterogeneity and are predominantly of the IgG1 and IgG2 subclasses.

A 22-amino acid synthetic peptide (P peptide) containing the conserved, shared autoantigenic determinants of the ribosomal P proteins was conjugated to rabbit serum albumin and used to analyze anti-P heterogeneity in 17 lupus sera. Anti-P peptide antibodies demonstrated moderate restriction in isotype (IgM and IgG, but not IgA), subclass (predominantly IgG1 and IgG2), light chain type (predominantly kappa) and spectrotype. In one serum, almost exclusive use of IgG2 and the kappa light chain was observed. These findings indicate that there is a nonrandom selection of heavy and light chain constant region genes as well as limited variable region diversity in the anti-P peptide response.

Autoantibodies↗
PubMed1987

Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.

S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 45 degrees C express HS genes in vitro approximately 8 times better than extracts from cells at 33 degrees C. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% of that seen in extracts from cells at 33 degrees C. These results correlate well with the levels of HS sigma factor and normal sigma factor bound to RNA polymerase. Thus, there was an 8-fold increase in the HS sigma factor and a 60% decrease in the normal sigma factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS sigma factor could be accounted for by a 3-fold increase in the level of HS sigma factor mRNA during heat induction.

DNA-Directed RNA Polymerases↗
PubMed1986

Identification and chemical synthesis of a ribosomal protein antigenic determinant in systemic lupus erythematosus.

The characteristics of eukaryotic ribosomal proteins P0, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE). P0, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the presence of 6 M urea. The apparent molecular size of the complex was 140 kDa as determined by gel filtration on a Sephadex G-200 column. P0 may, therefore, be the eukaryotic equivalent of Escherichia coli ribosomal protein L10. In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and P0 and P1 were found to be more acidic than their ribosome-bound counterparts. Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia salina (eL12). Sixteen SLE sera containing antibodies to P0, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 amino acids. Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins. These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).

Amino Acid Sequence↗
PubMed1986

In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes.

In Escherichia coli, the ability to elicit a heat shock response depends on the htpR gene product. Previous work has shown that the HtpR protein serves as a sigma factor (sigma 32) for RNA polymerase that specifically recognizes heat shock promoters (A.D. Grossman, J.W. Erickson, and C.A. Gross Cell 38:383-390, 1984). In the present study we showed that sigma 32 synthesized in vitro could stimulate the expression of heat shock genes. The in vitro-synthesized sigma 32 was found to be associated with RNA polymerase. In vivo-synthesized sigma 32 was also associated with RNA polymerase, and this polymerase (E sigma 32) could be isolated free of the standard polymerase (E sigma 70). E sigma 32 was more active than E sigma 70 with heat shock genes; however, non-heat-shock genes were not transcribed by E sigma 32. The in vitro expression of the htpR gene required E sigma 70 but did not require E sigma 32.

Chromatography, Gel↗
PubMed1985

Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes.

The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine gamma-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems. In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet). In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (approximately 40-60%) metJ gene expression. Thus, the metJ gene can be partially autoregulated by its gene product.

Bacterial Proteins↗
PubMed1985

Cloning and expression of the metE gene in Escherichia coli.

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.

Bacteriophage lambda↗
PubMed1985

Regulation of methionine synthesis in Escherichia coli: Effect of metJ gene product and S-adenosylmethionine on the expression of the metF gene.

The regulation of the expression of the Escherichia coli metF gene, which codes for 5,10-methylenetet-rahydrofolate reductase (EC 1.1.99.15), has been investigated by using a simplified DNA-directed in vitro system that measures the formation of the first dipeptide (fMet-Ser) of the gene product. The synthesis of fMet-Ser directed by a plasmid containing the metF gene is specifically inhibited by metJ protein (repressor protein). S-Adenosylmethionine enhances the inhibition by the metJ protein of metF gene expression. The inhibition by the metJ protein is at the level of transcription and the results suggest that S-adenosylmethionine is functioning as an allosteric effector.

Journal Article↗
PubMed1985

Light regulation of the synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase in peas: Evidence for translational control.

The specific activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) increases 30- to 50-fold when dark-grown pea seedlings are shifted into the light. The large subunit (LS) of this multimeric protein is known to be synthesized in the chloroplast, but plastids from dark-grown cells contain relatively low levels of LS. However, despite the low level of LS synthesis in the plastids of dark-grown plants, these organelles contain significant levels of LS mRNA. Hybridization studies showed that the amount of LS mRNA increased about 3-fold, relative to total plant RNA, when dark-grown plants were illuminated. This increase in LS mRNA can be accounted for by a similar increase in chloroplast genome copy number. It was found that the amount of translatable LS mRNA per mug of plastid RNA is similar when isolated from either dark-grown plants or dark-grown plants subjected to light. These results suggest that although light can increase the level of LS mRNA by increasing the copy number of this gene, the primary regulation of LS synthesis by light in pea chloroplasts is at the level of translation.

Journal Article↗
PubMed1984

Transcriptional activity of isolated maize chloroplasts.

Chloroplasts and etioplasts, isolated from light- or dark-grown Zea mays plants, respectively, can incorporate labeled UTP into RNA in a reaction stimulated by light or ATP. This in organello RNA synthesis proceeded at a linear rate for up to 2 h. When expressed per unit protein, plastids from dark-grown plants incorporated more UTP than those from light-grown plants, and the highest rate of UTP incorporation was found in plastids from light-stimulated leaves (grown previously in the dark). The in organello newly synthesized RNA was heterodispersed, with most transcripts smaller than 14 S. Specific transcripts were detected in organelles from both dark- and light-grown plants that contain sequences that are homologous to the mRNAs for the rbcL gene (coding for the large subunit of ribulose bisphosphate carboxylase (LS-RuBPCase] and for the psbA gene (32-kDa thylakoid membrane protein). Qualitatively, the newly synthesized in organello transcripts were similar from the dark and light organelles.

Adenosine Triphosphate↗
PubMed1984

Two initiation sites detected in the small s1 species of reovirus mRNA by dipeptide synthesis in vitro.

Reovirus mRNAs directed the synthesis of fMet dipeptides in a translation initiation system reconstituted from rabbit reticulocyte initiation and elongation factors, Artemia salina 80S ribosomes, yeast fMet-tRNAiMet and Escherichia coli3H-labeled aminoacyl tRNAs. As predicted from the GC(U,G) codon that follows the 5'-proximal AUG in half of the viral mRNA species, fMet-Ala was the predominant dipeptide product obtained in response to a mixture of mRNAs or to the separated size classes of medium (m) and small (s) mRNA. The four individual small mRNA species each directed the synthesis of an fMet dipeptide that was consistent with the utilization of the 5'-proximal AUG for initiation. In addition to fMet-Asp, the s1 mRNA also directed fMet-Glu synthesis indicative of initiation in a second reading frame at the 5'-penultimate AUG. The tripeptide fMet-Glu-Tyr was also synthesized from s1 mRNA, which further verified this second initiation site. mRNAs containing 5'-terminal GpppG were 10-15% as active as the corresponding m7G-capped templates. The dipeptide assay provides a rapid method for determining initiation sites in individual mRNAs or in mixtures of mRNAs.

Amino Acid Sequence↗
PubMed1983

Translational control of the expression of the beta subunit gene of E. coli RNA polymerase.

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the in vitro synthesis of fMet-Val, the N-terminal dipeptide of the beta subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of beta, beta 1 synthesis is at the level of translation. L factor (nusA gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.

Bacterial Proteins↗