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J E Hearst

Publications and source records attributed to J E Hearst.

At least 37 records · Page 2Linked to original sources
PubMed1994

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli.

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, beta-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for beta-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.

Alkyl and Aryl Transferases↗
PubMed1994

Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer.

We extend the technique of fluorescence resonance energy transfer (FRET) by introducing a luminescent terbium chelate as a donor and an organic dye, tetramethylrhodamine, as an acceptor. The results are consistent with a Förster theory of energy transfer, provided the appropriate parameters are used. The use of lanthanide donors, in general, and this pair, in particular, has many advantages over more conventional FRET pairs, which rely solely on organic dyes. The distance at which 50% energy transfer occurs is large, 65 A; the donor lifetime is a single exponential and long (millisecond), making lifetime measurements facile and accurate. Uncertainty in the orientation factor, which creates uncertainty in measured distances, is minimized by the donor's multiple electronic transitions and long lifetime. The sensitized emission of the acceptor can be measured with little or no interfering background, yielding a > 25-fold improvements in the signal-to-background ratio over standard donor-acceptor pairs. These improvements are expected to make distances > 100 A measurable via FRET. We also report measurement of the sensitized emission lifetime, a measurement that is completely insensitive to total concentration and incomplete labeling.

Base Sequence↗
PubMed1994

Efficient anchoring of RNA polymerase in Escherichia coli during coupled transcription-translation of genes encoding integral inner membrane polypeptides.

While it has been known that supercoiling of the DNA template can be induced by transcription, the mechanism and the efficiency of this process in vivo is not fully understood. We report here that transcription of genes encoding 16 S rRNA, a stable RNA species, or cytoplasmic polypeptides leads to very little or no detectable DNA supercoiling even under the optimum conditions in Escherichia coli. This indicates that hydrodynamic drag on the transcription complex (including RNA polymerase, nascent RNA, ribosomes, and nascent polypeptides) is not sufficient to anchor RNA polymerase during coupled transcription-translation. On the other hand, transcription of membrane-associated genes encoding integral inner membrane or exported periplasmic polypeptides leads to apparent DNA supercoiling. Transcription of genes encoding integral inner membrane polypeptides leads to significantly greater anchoring of RNA polymerase than does transcription of genes encoding periplasmic polypeptides. This may reflect differences in the coupling of transcription-translation with membrane association during expression of these two classes of polypeptides. Evidence is further presented to suggest that the anchoring of RNA polymerase is probably achieved through the interaction of nascent polypeptides with the cytoplasmic surface of the inner membrane during coupled transcription-translation. Moreover, transcriptions of a membrane-associated gene can, under certain circumstances, induce topological anchoring of an RNA polymerase transcribing a neighboring gene that ordinarily is not membrane-associated. Finally, the potential biological consequences of our findings are discussed.

Bacterial Proteins↗
PubMed1994

Efflux pumps and drug resistance in gram-negative bacteria.

The outer membrane of Gram-negative bacteria can only slow down the influx of lipophilic inhibitors, and so these bacteria need active efflux pumps of broad specificity to survive. Pumps such as the Escherichia coli Acr system and its homologs make Gram-negative bacteria resistant to dyes, detergents and antibiotics.

Anti-Bacterial Agents↗
PubMed1994

Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides.

Carotenoids have two major functions in bacterial photosynthesis, photoprotection and accessory light harvesting. The genes encoding many carotenoid biosynthetic pathways have now been mapped and cloned in several different species, and the availability of cloned genes which encode the biosynthesis of carotenoids not found in the photosynthetic genus Rhodobacter opens up the possibility of introducing a wider range of foreign carotenoids into the bacterial photosynthetic apparatus than would normally be available by producing mutants of the native biosynthetic pathway. For example, the crt genes from Erwinia herbicola, a gram-negative nonphotosynthetic bacterium which produces carotenoids in the sequence of phytoene, lycopene, beta-carotene, beta-cryptoxanthin, zeaxanthin, and zeaxanthin glucosides, are clustered within a 12.8-kb region and have been mapped and partially sequenced. In this paper, part of the E. herbicola crt cluster has been excised and expressed in various crt strains of Rhodobacter sphaeroides. This has produced light-harvesting complexes with a novel carotenoid composition, in which the foreign carotenoids such as beta-carotene function successfully in light harvesting. The outcome of the combination of the crt genes in R. sphaeroides with those from E. herbicola has, in some cases, resulted in an interesting rerouting of the expected biosynthetic sequence, which has also provided insights into how the various enzymes of the carotenoid biosynthetic pathway might interact. Clearly this approach has considerable potential for studies on the control and organization of carotenoid biosynthesis, as well as providing novel pigment-protein complexes for functional studies.

Carotenoids↗
PubMed1993

Polymer models for interphase chromosomes.

The overall geometry of chromosomes in mammalian cells during interphase is analyzed. On scales larger than approximately 10(5) bp, a chromosome is modeled as a Gaussian polymer subjected to additional forces that confine it to a subvolume of the cell nucleus. An appropriate partial differential equation for the polymer Green's function leads to predictions for the average geometric length between two points on the chromosome. The model reproduces several of the experimental observations: (i) a square root dependence of average geometric distance between two marked chromosome locations on their genomic separation over genomic length scales from approximately 10(5) to approximately 10(6) bp; (ii) an approach of the geometric distance to a maximum value for still larger genomic separations of the two points; (iii) overall chromosome localization in subdomains of the cell nucleus, as suggested by fluorescent labeling of whole chromosomes and by radiobiological evidence. The model is also consistent with known properties of the 30-nm chromatin fiber. It makes a testable prediction: that for two markers a given number of base pairs apart on a given chromosome, the average geometric separation is larger if the configuration is near one end of the chromosome than if it is near the center of the chromosome.

Animals↗
PubMed1993

Early evolution of photosynthesis: clues from nitrogenase and chlorophyll iron proteins.

Chlorophyll (Chl) is often viewed as having preceded bacteriochlorophyll (BChl) as the primary photoreceptor pigment in early photosynthetic systems because synthesis of Chl requires one fewer enzymatic reduction than does synthesis of BChl. We have conducted statistical DNA sequence analyses of the two reductases involved in Chl and BChl synthesis, protochlorophyllide reductase and chlorin reductase. Both are three-subunit enzymes in which each subunit from one reductase shares significant amino acid identity with a subunit of the other, indicating that the two enzymes are derived from a common three-subunit ancestral reductase. The "chlorophyll iron protein" subunits, encoded by the bchL and bchX genes in the purple bacterium Rhodobacter capsulatus, also share amino acid sequence identity with the nitrogenase iron protein, encoded by nifH. When nitrogenase iron proteins are used as outgroups, the chlorophyll iron protein tree is rooted on the chlorine reductase lineage. This rooting suggests that the last common ancestor of all extant photosynthetic eubacteria contained BChl, not Chl, in its reaction center, and implies that Chl-containing reaction centers were a late invention unique to the cyanobacteria/chloroplast lineage.

Amino Acid Sequence↗
PubMed1993

Laser-induced protein-DNA cross-links via psoralen furanside monoadducts.

We have developed a technique for cross-linking DNA binding proteins to DNA using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source. Several DNA binding proteins (T7 RNA polymerase, UvrB, single-stranded DNA binding protein of Escherichia coli, T4 gp32, and RecA of E. coli) are shown to cross-link to single-stranded psoralen monoadducted DNA oligos differing in length and sequence. Increasing fluences of laser light on a fixed ratio of DNA/protein resulted in an increase in the yield of cross-links. Titration experiments were carried out to measure the apparent cross-linking constant (KappXL) for T7 RNA polymerase or UvrB to a monoadducted 24 mer DNA. The estimated values for the apparent cross-linking constant were in the range of (2-3) x 10(-7) M for both T7 RNA polymerase and UvrB. The efficiency of cross-linking was investigated as a function of the length of adducted DNA and also as a fraction of the total noncovalent binding of proteins of psoralenated DNAs. The results showed that in the cases of T7 RNA polymerase and UvrB cross-linking was more efficient with short oligos (8 and 19 mers) as compared to longer oligos (50 mer). A tryptic peptide of T7 RNA polymerase that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography. Mass spectrometry and amino acid composition of this peptide revealed that it originated from a region between residues 558 and 608 of the primary structure of T7 RNA polymerase. Two other peptides cross-linked to oligos were also purified. Repeated attempts to perform Edman sequencing of the peptide-DNA conjugates failed. Overall evidence indicates that photo-cross-linking of furanside monoadducts occurred at multiple sites on the proteins. We have shown that T7 RNA polymerase molecules in a ternary complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light. Evidence suggests that the arrested polymerase molecules existed in multiple conformations on the DNA template. This method of transcriptional cross-linking offers a new method for preparing highly stable elongation complexes for further studies.

Bacteriophage T7↗
PubMed1993

Molecular matchmakers.

Molecular matchmakers are a class of proteins that use the energy released from the hydrolysis of adenosine triphosphate to cause a conformational change in one or both components of a DNA binding protein pair to promote formation of a metastable DNA-protein complex. After matchmaking the matchmaker dissociates from the complex, permitting the matched protein to engage in other protein-protein interactions to bring about the effector function. Matchmaking is most commonly used under circumstances that require targeted, high-avidity DNA binding without relying solely on sequence specificity. Molecular matchmaking is an extensively used mechanism in repair, replication, and transcription and most likely in recombination and transposition reactions, too.

DNA↗
PubMed1993

DNA repair by eukaryotic nucleotide excision nuclease. Removal of thymine dimer and psoralen monoadduct by HeLa cell-free extract and of thymine dimer by Xenopus laevis oocytes.

Using a human cell-free extract, we have recently shown that thymine dimers are removed from DNA in oligonucleotides 27-29 nucleotides in length (Huang, J. C., Svoboda, D. L., Reardon, J. T., and Sancar, A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3664-3668). In this study we find that the excision reaction is dependent on ATP, the excised fragments range in length from 27-32 nucleotides, and have 5'-P and 3'-OH termini. We also found that a thymine-psoralen furan side monoadduct is excised from the DNA with a similar incision pattern, indicating that in humans bulky adducts are removed from DNA by the same enzyme system which hydrolyzes mainly the 22-24th and the 5th phosphodiester bonds 5' and 3', respectively, to the lesion. This incision pattern might be common to eukaryotic excision nucleases as thymine dimers were removed from DNA by the same dual incision pattern by Xenopus laevis oocytes.

Adenosine Triphosphate↗
PubMed1993

In vitro expression and activity of lycopene cyclase and beta-carotene hydroxylase from Erwinia herbicola.

The cyclisation of lycopene to beta-carotene and the hydroxylation of beta-carotene to zeaxanthin are common enzymatic steps in the biosynthesis of carotenoids in a wide range of bacteria, fungi, and plants. We have individually expressed in E. coli the two genes coding for these enzymatic steps in Erwinia herbicola. The cyclase and hydroxylase enzymes have apparent molecular weights of 43 kDa and 22 kDa, respectively, as determined by SDS-PAGE. Hydroxylase in vitro activity was obtained only in the cytoplasmic fraction. Cyclase also demonstrated enzyme activity in a crude cell-free lysate, although to a lesser extent.

Base Sequence↗
PubMed1993

Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation.

The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

Aerobiosis↗
PubMed1993

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.

Acriflavine↗
PubMed1993

Analysis of the promoter and regulatory sequences of an oxygen-regulated bch operon in Rhodobacter capsulatus by site-directed mutagenesis.

The biosynthesis of pigments (carotenoids and bacteriochlorophylls) in the photosynthetic bacterium Rhodobacter capsulatus is regulated by the oxygen concentration in the environment. However, the mechanism of this regulation has remained obscure. In this study, transcriptional fusions of the bchCXYZ promoter region to lacZ were used to identify the promoter and regulatory sequences governing transcription of these bacteriochlorophyll biosynthesis genes. The promoter region was identified in vivo by making deletions and site-directed mutations. The 50 bp upstream of the promoter region was shown to be required for the oxygen-dependent transcriptional regulation of bchCXYZ. A previously described palindrome sequence is also likely involved in the regulation. A gel mobility shift assay further defined the interaction of transcription regulators with these DNA sequence elements in vitro and demonstrated that a DNA-protein complex is formed at this promoter region. Since the suggested promoter sequence and the palindrome sequence are found upstream of several other bch and crt operons, these sequences may be responsible for regulating oxygen-dependent pigment biosynthesis at the level of transcription in R. capsulatus. In addition, these cis-acting DNA elements are not found upstream of puh and puf operons, which encode the structural polypeptides of the reaction center and light-harvesting I complexes. This observation supports the model of different regulatory mechanism for the pigment biosynthesis enzymes and structural polypeptides required for the production of the photosynthetic apparatus.

Bacteriochlorophylls↗
PubMed1993

The Rhodobacter capsulatus chlorin reductase-encoding locus, bchA, consists of three genes, bchX, bchY, and bchZ.

The bchA locus of Rhodobacter capsulatus codes for the chlorin reductase enzyme in the bacteriochlorophyll synthesis pathway. Previous work has suggested that this locus might encompass a single gene. We have sequenced the bchA locus and found it to contain three coding segments, which we designate bchX, bchY, and bchZ. Each coding segment contains its own translational initiation sequence and follows codon utilization patterns consistent with those of previously published R. capsulatus genes. When various regions of the bchA locus and flanking sequences were subcloned into an expression vector and expressed in Escherichia coli, the three coding segments were all expressed as separate peptides. Finally, conservation of amino acid sequences between bchX and a subunit of the protochlorophyllide reductase (bchL, 34% identity) and the nitrogenase Fe protein (nifH, 30 to 37% identity) suggests structural and mechanistic commonalities among all three proteins.

Amino Acid Sequence↗