PubMed Health⌕ Search

Biomedical subjects

H Weissbach

Publications and source records attributed to H Weissbach.

At least 73 records · Page 4Linked to original sources
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

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

Escherichia coli integration host factor inhibits the NusA stimulation of RNA polymerase sigma subunit synthesis in vitro.

As reported previously, Integration Host Factor (IHF) stimulates cII expression but the stimulatory effect is prevented by the NusA protein (Peacock and Weissbach, 1985, Biochem. Biophys. Res. Commun. 127, 1026-1031). The interaction between IHF and the NusA protein has been investigated further in studies on the in vitro expression of the genes for the beta (rpoB) and sigma (rpoD) subunits of RNA polymerase, both known to be stimulated by NusA. The NusA stimulation of rpoD expression can be prevented by IHF, but IHF has no effect by itself on rpoD expression. IHF does not influence rpoB expression either in the presence or absence of NusA.

Bacterial Proteins↗
PubMed1985

IHF stimulation of lambda cII gene expression is inhibited by the E. coli NusA protein.

The effects of E. coli proteins Integrative Host Factor (IHF) and NusA on the regulation of lambda cII gene expression are presented. As reported previously (Peacock et al. [1984] Proc. Natl. Acad. Sci. USA 81, 6009-6013), IHF stimulates the DNA-directed in vitro synthesis of cII protein or its first dipeptide, fMet-Val. Whereas NusA, by itself, has no effect on cII expression, the presence of NusA inhibits the IHF-mediated stimulation of cII synthesis.

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

In vitro stimulation of Escherichia coli RNA polymerase sigma subunit synthesis by NusA protein.

A simplified DNA-directed in vitro system which measures synthesis of the NH2-terminal dipeptides of gene products has been used to study the expression of rpoD, the gene coding for the sigma subunit of Escherichia coli RNA polymerase. The rpoD gene is part of a complex operon which also includes the genes for ribosomal protein S21 (rpsU) and primase (dnaG). Primary promoters have been identified upstream of the structural genes, but there are secondary (internal) promoters within the dnaG gene that are involved in the expression of rpoD. Significant expression of the rpsU and rpoD genes was observed in the in vitro dipeptide system using plasmid pBS105, which contains both external and internal promoters. With plasmid pMRG-1, which contains only the internal promoters, only rpoD expression was observed. From either template, synthesis of the NH2-terminal dipeptide of sigma, fMet-Glu, is stimulated about threefold by the E. coli nusA gene product. In addition, NusA protein stimulates synthesis of the entire sigma protein in a defined in vitro system. NusA protein has no effect on the expression of the upstream gene rpsU, and the stimulation of rpoD expression by NusA protein is at the level of transcription. The results are consistent with the known role of NusA protein in modulating transcription at pause or attenuation sites.

Bacterial Proteins↗
PubMed1985

An in vitro system to measure gene expression based on dipeptide synthesis.

A simplified E. coli in vitro system has been developed to study gene expression based on the synthesis of the first di- or tripeptide of the gene product. Plasmids containing bacterial and chloroplast genes have been used as templates in this system. The expression of the E. coli L10 operon, which is under both transcriptional and translational control, has been investigated in some detail using the dipeptide system. A similar system has been developed, using eukaryotic translation components, to measure the expression of eukaryotic mRNA based on dipeptide formation.

Animals↗
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

In vitro expression and characterization of the translation start site of the psbA gene product (QB protein) from higher plants.

The psbA gene from higher plants, which codes for the atrazine herbicide binding protein of photosystem II (QB protein), has been recently sequenced by various laboratories. From these data there are two potential translation sites, one yielding a protein of 38,500 kd and another a protein of 34,500 kd. In the present study, cloned psbA gene sequences from maize, tobacco, and pea have been expressed in a highly defined E. coli in vitro transcription/translation system. In order to determine the start site of translation, we also have employed a simplified E. coli system designed to synthesize the first di- or tripeptide of the gene product. From these results, it is clear that the first ATG of the longest open reading frame of the psbA gene, that begins fMet-Thr, is not recognized in vitro. Instead, the next downstream Met at position 37 is the initiation site, since the expected dipeptide fMet-Ile is synthesized from all psbA clones. These data are in accord with the in vivo results that the gene product is a precursor protein of 34,500 kd.

Chloroplasts↗