Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Isolation and characterization of the membrane-bound cytochrome c-554 from the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus

The membrane-bound photooxidizable cytochrome c-554 from Chloroflexus aurantiacus has been purified. The purified protein runs as a single heme staining band on SDS-PAGE with an apparent molecular mass of 43 000 daltons. An extinction coefficient of 28 ± 1 mM^–1 cm^–1 per heme at 554 nm was found for the dithionite-reduced protein. The potentiometric titration of the hemes takes place over an extended range, showing clearly that the protein does not contain a single heme in a well-defined site. The titration can be fit to a Nernst curve with midpoint potentials at 0, +120, +220 and +300 mV vs the standard hydrogen electrode. Pyridine hemochrome analysis combined with a Lowry protein assay and the SDS-PAGE molecular weight indicates that there are a minimum of three, and probably four hemes per peptide. Amino acid analysis shows 5 histidine residues and 29% hydrophobic residues in the protein. This cytochrome appears to be functionally similar to the bound cytochrome from Rhodopseudomonas viridis. Both cytochrome c-554 from C. aurantiacus and the four-heme cytochrome c-558-553 from R. viridis appear to act as direct electron donors to the special bacteriochlorophyll pair of the photosynthetic reaction center. They have a similar content of hydrophobic amino acids, but differ in isoelectric point, thermodynamic characteristics, spectral properties, and in their ability to be photooxidized at low temperature.Abbreviations LDAO lauryl dimethyl amine-N-oxide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - mV millivolt - E_m.8 midpoint potential at pH 8.0 - ODV optical density x volume in ml     

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl₄, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at 747 nm.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGGipm0dc9vqaqpepu0xbbG8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeq4Xdm2aaW% baaSqabeaacaaIYaaaaaaa!3777!\[\chi ^2 \] chi-square - FWHM full-width at half-maximum     

Competition of cyclooctenes and cyclooctadienes for ethylene binding and activity in plants

trans-Cyclooctene, cis,trans-1,5-cyclooctadiene, and cis,trans-1,3-cyclooctadiene have been compared with the cis and cis,cis isomers and with 2,5-norbornadiene for competition with ethylene for binding in mung bean sprouts and tobacco and for action (induction of chlorophyll degradation) in banana. The compounds containing a trans double bond were much more effective in competition for binding and action than the cis and cis,cis compounds. trans-Cyclooctene and cis,trans-1,3-cyclooctadiene were in the general range of 50–90 times more effective than 2,5-norbornadiene.R.J. Reynolds Research Apprentice     

P48-Triggered transmembrane signaling transduction of human monocytes:mobilization of calcium ion and activation of protein kinase C(PKC)

INTRODUCTI0NThedifferentiati0nofcelIsalongthemonocyte-macr0phagepathwayandthesig-nalsinvo1vedinthesecel1sacquiringtheabilitytokilltum0rcellsarenotfllllyundersto0d.Wehavebeenstudingamoleculewhichappearst0beanimportantmemberofthecytokinenetworkinvo1vedintheregulati0nmonocyteactivation.ThiscytokinetermedP48wasisolatedfr0mthehllmannullcellleukemiacell1ineReh.IthasbeenpurifiedtohomogeneityandfOundtobedistinctfrominterferongamma,col0nystimulatingfactors(CSFs)andTNFalphaalldbeta[1,2].Func-ti…     

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leechHaementeria ghilianii.In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an in-frame fusion of the ghilanten-coding sequences with the region encoding the pre-pro α-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter.Pichia pastorisyeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5′-AOX1locus via homologous recombination. Both strains yielded His⁺transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level ofr-ghilanten than KM 71. Significant clonal variation in the expression ofr-ghilanten was found among the His⁺transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales.r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.     

Protein structure,electron transfer and evolution of prokaryotic photosynthetic reaction centers

Photosynthetic reaction centers from a variety of organisms have been isolated and characterized. The groups of prokaryotic photosynthetic organisms include the purple bacteria, the filamentous green bacteria, the green sulfur bacteria and the heliobacteria as anoxygenic representatives as well as the cyanobacteria and prochlorophytes as oxygenic representatives. This review focuses on structural and functional comparisons of the various groups of photosynthetic reaction centers and considers possible evolutionary scenarios to explain the diversity of existing photosynthetic organisms.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - Rb Rhodobacter - Rp Rhodopseudomonas     

Spectral heterogeneity and time-resolved spectroscopy of excitation energy transfer in membranes of Heliobacillus mobilis at low temperatures.

Transient absorption difference spectra in the Qy absorption band from membranes of Heliobacillus mobilis were recorded at 140 and 20 K upon 200 fs laser pulse excitation at 590 nm. Excitation transfer from short wavelength absorbing forms of bacteriochlorophyll g to long wavelength bacteriochlorophyll g occurred within 1-2 ps at both long wavelength bacteriochlorophyll g occurred within 1-2 ps at both temperatures. In addition, a slower energy transfer process with a time constant of 15 ps was observed at 20 K within the pool of long wavelength-absorbing bacteriochlorophyll g. Energy transfer from long wavelength antenna pigments to the primary electron donor P798 was observed, yielding the primary charge-separated state P798+A0-. The time constant for this process was 30 ps at 140 K and about 70 ps at 20 K. A decay component with smaller amplitude and a lifetime of up to hundreds of picoseconds was observed that was centered around 814 nm at 20 K. Kinetic simulations using simple lattice models reproduce the observed decay kinetics at 295 and 140 K, but not at 20 K. The kinetics of energy redistribution within the spectrally heterogeneous antenna system at low temperature argue against a simple "funnel" model for the organization of the antenna of Heliobacillus mobilis and favor a more random spatial distribution of spectral forms. However, the relatively high rate of energy transfer from long wavelength antenna bacteriochlorophyll g to the primary electron donor P798 at low temperature is difficult to explain with either of these models.     

Excited state dynamics in photosystem I: effects of detergent and excitation wavelength.

Femtosecond transient absorption spectroscopy has been used to investigate the energy transfer and trapping processes in both intact membranes and purified detergent-isolated particles from a photosystem II deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803, which contains only the photosystem I reaction center. Processes with similar lifetimes and spectra are observed in both the membrane fragments and the detergent-isolated particles, suggesting little disruption of the core antenna resulting from the detergent treatment. For the detergent-isolated particles, three different excitation wavelengths were used to excite different distributions of pigments in the spectrally heterogeneous core antenna. Only two lifetimes of 2.7-4.3 ps and 24-28 ps, and a nondecaying component are required to describe all the data. The 24-28 ps component is associated with trapping. The trapping process gives rise to a nondecaying spectrum that is due to oxidation of the primary electron donor. The lifetimes and spectra associated with trapping and radical pair formation are independent of excitation wavelength, suggesting that trapping proceeds from an equilibrated excited state. The 2.7-4.3 ps component characterizes the evolution from the initially excited distribution of pigments to the equilibrated excited state distribution. The spectrum associated with the 2.7-4.3 ps component is therefore strongly excitation wavelength dependent. Comparison of the difference spectra associated with the spectrally equilibrated state and the radical pair state suggests that the pigments in the photosystem I core antenna display some degree of excitonic coupling.