Seasonal variations in the levels of PAH-DNA adducts in young adults living in Mexico City.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous components of polluted air. The Mexico City Metropolitan Area (MCMA), one of the most densely populated areas in the world, is 2240 m above sea level. At this altitude, less oxygen is available, making combustion less efficient and therefore producing more PAH pollutants. According to the Automatic Monitoring Network in Mexico City (RAMA, for its Spanish initials; http://www.sma.df.gob.mx/simat2/informaciontecnica/index.php?opcion=5&opciondifusion_bd=90), which performs environmental monitoring, the critical air pollutants in Mexico City are ozone and particulate matter (PM). PM emissions increase during the dry season (winter to spring) and decrease during the rainy season (summer to autumn). The bioactivation of some PAHs produces reactive metabolites that bind to DNA, and the presence of elevated levels of PAH-DNA adducts in tissues such as blood lymphocytes represents an elevated risk for the development of cancer. We have compared the levels of PAH-DNA adducts and the percentage of cells with chromosomal aberrations (CWAs) using a matched set of peripheral blood lymphocytes obtained on two separate occasions from young non-smoking inhabitants of the MCMA (n = 92) during the 2006 dry season and the following rainy season. PAH-DNA adducts were analysed using the r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay (CIA). The percentages of CWA were determined in cultured lymphocytes from the same individuals. Both DNA adduct levels and chromosomal aberrations were tested for correlation with lifestyle and the polymorphisms of cytochromes P450 CYP1A1 and CYP1B1 as well as glutathione-S-transferases GSTM1 and GSTT1. The levels of PAH-DNA adducts were significantly higher (P < 0.001) in the dry season (10.66 ± 3.05 per 10(9) nt, n = 92) than during the rainy season (9.50 ± 2.85 per 10(9) nt, n = 92) and correlated with the seasonal levels of particulate matter with a diameter of ≤ 10 µm (PM(10)). The percentage of CWA was not seasonally related; however, significant associations between the number of risk alleles and adduct levels in the dry (R = 0.298, P = 0.048) and in the wet seasons (R = 0.473, P = 0.001) were observed.

cDNA sequences for transcription factors and signaling proteins of the hemichordate Saccoglossus kowalevskii: efficacy of the expressed sequence tag (EST) approach for evolutionary and developmental studies of a new organism.

We describe a collection of expressed sequence tags (ESTs) for Saccoglossus kowalevskii, a direct-developing hemichordate valuable for evolutionary comparisons with chordates. The 202,175 ESTs represent 163,633 arrayed clones carrying cDNAs prepared from embryonic libraries, and they assemble into 13,677 continuous sequences (contigs), leaving 10,896 singletons (excluding mitochondrial sequences). Of the contigs, 53% had significant matches when BLAST was used to query the NCBI databases (< or = 10(-10)), as did 51% of the singletons. Contigs most frequently matched sequences from amphioxus (29%), chordates (67%), and deuterostomes (87%). From the clone array, we isolated 400 full-length sequences for transcription factors and signaling proteins of use for evolutionary and developmental studies. The set includes sequences for fox, pax, tbx, hox, and other homeobox-containing factors, and for ligands and receptors of the TGFbeta, Wnt, Hh, Delta/Notch, and RTK pathways. At least 80% of key sequences have been obtained, when judged against gene lists of model organisms. The median length of these cDNAs is 2.3 kb, including 1.05 kb of 3' untranslated region (UTR). Only 30% are entirely matched by single contigs assembled from ESTs. We conclude that an EST collection based on 150,000 clones is a rich source of sequences for molecular developmental work, and that the EST approach is an efficient way to initiate comparative studies of a new organism.

Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato.

We show that phytochromes modulate differentially various facets of light-induced ripening of tomato fruit (Solanum lycopersicum L.). Northern analysis demonstrated that phytochrome A mRNA in fruit accumulates 11.4-fold during ripening. Spectroradiometric measurement of pericarp tissues revealed that the red to far-red ratio increases 4-fold in pericarp tissues during ripening from the immature-green to the red-ripe stage. Brief red-light treatment of harvested mature-green fruit stimulated lycopene accumulation 2. 3-fold during fruit development. This red-light-induced lycopene accumulation was reversed by subsequent treatment with far-red light, establishing that light-induced accumulation of lycopene in tomato is regulated by fruit-localized phytochromes. Red-light and red-light/far-red-light treatments during ripening did not influence ethylene production, indicating that the biosynthesis of this ripening hormone in these tissues is not regulated by fruit-localized phytochromes. Compression analysis of fruit treated with red light or red/far-red light indicated that phytochromes do not regulate the rate or extent of pericarp softening during ripening. Moreover, treatments with red or red/far-red light did not alter the concentrations of citrate, malate, fructose, glucose, or sucrose in fruit. These results are consistent with two conclusions: (a) fruit-localized phytochromes regulate light-induced lycopene accumulation independently of ethylene biosynthesis; and (b) fruit-localized phytochromes are not global regulators of ripening, but instead regulate one or more specific components of this developmental process.

The phytochrome gene family in tomato and the rapid differential evolution of this family in angiosperms.

A reexamination of the genome of the tomato (renamed Solanum lycopersicum L.) indicates that it contains five, or at most perhaps six, phytochrome genes (PHY), each encoding a different apoprotein (PHY). Five previously identified tomato PHY genes have been designated PHYA, PHYB1, PHYB2, PHYE, and PHYF. A molecular phylogenetic analysis is consistent with the hypothesis that the angiosperm PHY family is composed of four subfamilies (A, B, C/F, and E). Southern analyses indicate that the tomato genome does not contain both a PHYC and a PHYF. Molecular phylogenetic analyses presented here, which utilize for the first time full-length PHY sequences from two completely characterized angiosperm gene families, indicate that tomato PHYF is probably an ortholog of Arabidopsis PHYC. They also confirm that the angiosperm PHY family is undergoing relatively rapid differential evolution. Assuming PHYF is an ortholog of PHYC, PHY genes in eudicots are evolving (Ka/site) at 1.52-2.79 times the rate calculated as average for other plant nuclear genes. Again assuming PHYF is an ortholog of PHYC, the rate of evolution of the C and E subfamilies is at least 1.33 times the rate of the A and B subfamilies. PHYA and PHYB in eudicots are evolving at least 1.45 times as fast as their counterparts in the Poaceae. PHY functional domains also exhibit different evolutionary rates. The C-terminal region of angiosperm PHY (codons 800-1105) is evolving at least 2.11 times as fast as the photosensory domain (codons 200-500). The central region of a domain essential for phytochrome signal transduction (codons 652-712) is also evolving rapidly. Nonsynonymous substitutions occur in this region at 2.03-3.75 times the average rate for plant nuclear genes. It is not known if this rapid evolution results from selective pressure or from the absence of evolutionary constraint.

Characterization of the gene encoding the apoprotein of phytochrome B2 in tomato, and identification of molecular lesions in two mutant alleles.

The structure of the gene encoding the apoprotein of tomato phytochrome B2 (PHYB2) has been determined from genomic and cDNA sequences. The coding region is organized into four exons, like almost every other angiosperm phytochrome (phy). The deduced phyB2 apoprotein (PHYB2) consists of 1121 amino acids, with 82, 74 and 70% identity to tomato PHYB1, Arabidopsis PHYB, and Arabidopsis PHYD, respectively. In order to facilitate the identification of new mutants, we constructed a double mutant that is deficient in phyA and phyB1. When grown in white light, this mutant becomes only slightly taller than wild type and is similar in phenotype to the monogenic phyB1-deficient mutant. This double mutant has been used as the parent line for mutagenesis with gamma radiation. Several recessive mutants with long hypocotyls and reduced anthocyanin content were selected under white light and screened for mutations in PHYB2, PHYE and PHYF. Two of the triple-mutant lines, designated 55H and 70F, had elongated hypocotyls and fruit trusses, and pale immature fruits. Both belong to the same complementation group and both were found to have defects in PHYB2. Line 70F was found by Northern analysis to have a slightly larger PHYB2 transcript. Part or all of the intron between the second and third exons was found to be retained following RT-PCR of PHYB2 mRNA from line 70F. Three base substitutions were detected near the donor splice site for this intron, including a change from the consensus /GT to /GA at the 5' end of this intron. In every case, the C-terminal 164 amino acids of PHYB2 were replaced by 59 nonsense amino acids followed by a stop codon. Sequencing of PHYB2 from 55H revealed a single-nucleotide deletion near the end of the third exon, resulting in one incorrect codon followed immediately by a stop codon. The predicted mutant apoprotein in 55H is 90 residues shorter than wild-type PHYB2.

Characterization of tomato PHYB1 and identification of molecular defects in four mutant alleles.

The structure of the gene encoding the apoprotein of phytochrome B (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcription start site was found by 5' RACE at -116. Tomato PHYB1 spans 7 kb starting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino acids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% and 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse transcriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the coding region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily red-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each mutation arose from a different, single-base substitution. Allele tri1 is presumably a null because the mutation introduces a stop at codon 92. In tri3, val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri1. Alleles tri2 and tri4 encode proteins truncated at their C-termini. The former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lacks 225.

Molecular analysis of PHYA in wild-type and phytochrome A-deficient mutants of tomato.

Tomato (Lycopersicon esculentum Mill., recently redesignated Solanum lycopersicum L.), an agronomically important crop plant, has been adopted as a model species complementary to Arabidopsis in which to characterize the phytochrome family. Here we describe the cloning and molecular characterization of the gene encoding the apoprotein of phytochrome A in wild-type tomato and in the far-red-light-insensitive (fri1 and fri2) tomato mutants. The physical organization of this gene is similar to that of other angiosperm phytochromes with the four exons of the coding region interrupted by three introns. The pool of transcripts is heterogeneous due to multiple transcription start sites and to three modes of alternative splicing of the 5' leader. The leader in each alternative transcript carries multiple upstream open reading frames of considerable length. At the genomic level, both fri mutants share an identical base substitution which changes a consensus AG/ to TG/ at the 3' end of the intron between exons 1 and 2. This mutation leads to aberrant processing of the resultant pre-mRNA. While most mature transcripts retain the mutated intron, both cryptic splicing and exon skipping were also detected. Cryptic splicing occurred both upstream and downstream from the wild-type splice site. These observations are consistent with the hypothesis that exon definition in splicing of plant pre-mRNAs plays a secondary role to that of intron definition. Analysis of the frequency with which potentially functional phytochrome A apoproteins might be produced indicates that both fri1 and fri2 have less than 1% of the wild-type phytochrome A level.

Temporal and photoregulated expression of five tomato phytochrome genes.

Quantitative measurements of the absolute amounts of mRNAs transcribed from each of five phytochrome genes (PHYA, PHYB1, PHYB2, PHYE, PHYF) throughout the life cycle of a tomato plant and in response to changes in ambient light conditions are reported here. From their lowest level in unimbibed seed, all five transcripts increase by from 10- to 1000-fold during the first 24 h following the onset of imbibition, both in continuous darkness and in a greenhouse. In a greenhouse and on a whole-plant basis, all continue to increase throughout day 6, after which all but PHYE decline over the next week to a plateau at about one-half of the maximal value. PHYE mRNA differs in that in continues to increase in abundance during the first 2-3 weeks and thereafter remains at that maximal level. In adult plants, on a whole-plant basis and in decreasing order of abundance, PHYA, PHYB1, PHYE, PHYB2, and PHYF transcripts were present at approximately 120, 40, 40, 15, and 8 mumol microgram-1 of poly(A)(+)-enriched RNA, respectively. The data are consistent with the demonstrated roles of phytochromes A and B1 during seedling development and lead to the suggestion that phytochrome E might have a more important role in mature plants. Somewhat unexpectedly, PHYA and PHYB2 expression patterns are very similar. In seedlings, PHYA and PHYB2 exhibit the greatest increase in expression following a light-to-dark transition, as well as the greatest decrease following a dark-to-light transition. PHYA and PHYB2 are also similar in that both exhibit comparable variation on a natural diurnal cycle, while PHYB1 also exhibits variation but with a markedly different phase. The diurnal variation in expression of PHYA, PHYB1 and PHYB2 is consistent with the possibility that one or more of the phytochromes they encode is important with respect not only to photoperiodic behavior but also to the regulation of other events whose photosensitivity varies during a diurnal cycle.

Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies.

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate-zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.

The sorghum photoperiod sensitivity gene, Ma3, encodes a phytochrome B.

The Ma3 gene is one of six genes that regulate the photoperiodic sensitivity of flowering in sorghum (Sorghum bicolor [L.] Moench). The ma3R mutation of this gene causes a phenotype that is similar to plants that are known to lack phytochrome B, and ma3 sorghum lacks a 123-KD phytochrome that predominates in light-grown plants and that is present in non-ma3 plants. A population segregating for Ma3 and ma3 was created and used to identify two randomly amplified polymorphic DNA markers linked to Ma3. These two markers were cloned and mapped in a recombinant inbred population as restriction fragment length polymorphisms. cDNA clones of PHYA and PHYC were cloned and sequenced from a cDNA library prepared from green sorghum leaves. Using a genome-walking technique, a 7941-bp partial sequence of PHYB, was determined from genomic DNA from ma3 sorghum. PHYA, PHYB, and PHYC all mapped to the same linkage group. The Ma3-linked markers mapped with PHYB more than 121 centimorgans from PHYA and PHYC. A frameshift mutation resulting in a premature stop codon was found in the PHYB sequence from ma3 sorghum. Therefore, we conclude that the Ma3 locus in sorghum is a PHYB gene that encodes a 123-kD phytochrome.

Absolute quantification of five phytochrome transcripts in seedlings and mature plants of tomato (Solanum lycopersicum L.).

Described here are the first quantitative measurements of absolute amounts of mRNAs transcribed from individual members of a phytochrome gene (PHY) family. The abundances of PHY mRNAs were determined for dry seed and for selected organs of green-house-grown tomato (Solanum lycopersicum L.) seedlings and mature plants. With a Phosphoimager, absolute amounts of PHYA, PHYB1, PHYB2, PHYE and PHYF transcripts were measured with reference to standard curves prepared from mRNA fragments synthesized in vivo. Methodology was developed permitting the use of polymerase chain reaction (PCR)-generated probes derived from a highly conserved region of PHY, obviating the necessity to clone cDNAs and to isolate probes derived from their 3' non-coding regions. In dry seeds, PHYB1 mRNA appeared to be most abundant (4-5 mumol/mol mRNA) while in all other instances PHYA mRNA predominated. In seedlings, PHYB1, PHYB2, PHYE, and PHYF mRNAs were most abundant in the shoot (25-87 mumol/mol mRNA) while PHYA mRNA was most abundant in the root (325 mumol/mol mRNA). In adult plants, the levels of PHYA. PHYB1 and PHYE mRNAs were relatively uniform among different organs (approx. 100, 75, and 10 mumol/mol mRNA, respectively). In contrast, PHYB2 and PHYF were expressed preferentially in ripening fruits (35 and 47 mumol/mol mRNA, respectively), indicative of a possible role in fruit ripening for the phytochromes they encode. In general, the order of decreasing abundance of the five mRNAs for both seedlings and mature plants was PHYA, PHYB1, PHYE, PHYB2 and PHYF. Based upon observations that relatively modest changes in the extent of PHY expression result in changes in phenotype, the differential expression of each of the five tomato PHY described here is predicted to impact upon the spatial expression of biological activity of each phytochrome.

The mapping of phytochrome genes and photomorphogenic mutants of tomato.

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ( j )) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1.

The phytochrome gene family in tomato includes a novel subfamily.

Data presented here define five tomato phytochrome genes (PHY) and indicate the existence of additional PHY in the tomato genome. Portions of each gene, encoding amino acids 203 through 315 in a consensus amino acid sequence, were amplified by polymerase chain reaction. Four of these genes, PHYA, PHYB1, PHYB2 and PHYE, are members of previously identified PHY subfamilies, while the fifth, PHYF, is identified as a member of a new PHY subfamily. PHYA, PHYB1, PHYB2 and PHYE fragments encode amino acid sequences that share 88% to 98% sequence identity with their Arabidopsis counterparts. The PHYF fragment, however, encodes a polypeptide that shares only 65% to 74% sequence identity with previously identified Arabidopsis phytochromes. A phylogenetic analysis suggests that PHYF arose soon after, or perhaps prior to, the origin of angiosperms. This analysis leads to the prediction that PHYF might be widespread among angiosperms, including both monocotyledons and dicotyledons. Each of the five tomato PHY is expressed as a transcript of sufficient size to encode a full-length phytochrome apoprotein. Two PHYF transcripts, 4.4 and 4.7 kb in length, have been detected in 9-day-old light-grown seedlings, consistent with either multiple transcription start sites or differential processing. Analyses of genomic Southern blots hybridized with radiolabelled RNA probes derived from the five tomato PHY, as well as Arabidopsis PHYC, indicate that the tomato genome contains as many as 9 to 13 PHY. The tomato PHY family is apparently not only different from, but also larger than, the PHY family presently described for Arabidopsis.

Tomato contains two differentially expressed genes encoding B-type phytochromes, neither of which can be considered an ortholog of Arabidopsis phytochrome B.

Tomato (Solanum lycopersicon L.) contains two B-type phytochrome genes (PHYB1 and PHYB2). Fragments of these two PHYB were cloned following amplification by the polymerase chain reaction of a portion of their relatively well conserved 5' coding regions. Polypeptides encoded by these gene fragments exhibit 90% sequence identity. These two PHYB are independently expressed in organ-specific fashion. In mature plants, PHYB2 mRNA is most abundant in fruit and PHYB1 mRNA in expanded leaves. A phylogenetic analysis fails to establish which tomato PHYB is orthologous to either Arabidopsis PHYB or PHYD, the latter being a second B-type phytochrome. Instead, this analysis indicates that following the divergence of the Solanaceae and Brassicaceae from one another, a PHYB gene duplicated independently in each lineage. Consequently, Arabidopsis PHYB mutants cannot be considered strictly equivalent to the tomato tri mutants, which appear to be mutated at the PHYB1 locus. Similarly, other putative PHYB mutants might not be equivalent to those described for Arabidopsis and tomato. This situation complicates efforts to determine 'PHYB function' because there might be no one answer to this question.

Severity index scores correlate with survival of AIDS patients.

A major concern of hospices treating AIDS patients is survival prognosis. Patients are eligible for government hospice benefits only if they are in the last six months of life, but for AIDS patients who present at different disease stages, it is often difficult to predict survival. We have tested an index of AIDS severity developed by Alemi et al. (1991, Interfaces, 21(3), 105) for its ability to predict survival in hospice-AIDS patients. Using retrospective analysis of medical records, a severity index (SI) score was determined for 26 AIDS patients who were admitted at different disease stages to a South Florida hospice. The length of stay for each patient was also recorded. The patients fell clearly into two groups, those with stays of six months or less and those with stays of more than six months. The mean SI scores of the two groups were .9188 and .7845, respectively. These scores were significantly different at the p = .005 level. In this preliminary study, the severity score correlated well with survival prognosis. Based on these results, it appears that the severity index may have great utility in predicting survival for AIDS patients seeking hospice admission.

Identification of two loci involved in phytochrome expression in Nicotiana plumbaginifolia and lethality of the corresponding double mutant.

Four Nicotiana plumbaginifolia mutants exhibiting long hypocotyls and chlorotic cotyledons under white light, have been isolated from M2 seeds following mutagenesis with ethyl methane sulphonate. In each of these mutants, this partly etiolated in white light (pew) phenotype is due to a recessive nuclear mutation at a single locus. Complementation analysis indicates that three mutants, dap5, ems28 and ems3-6-34, belong to a single complementation group called pew1, while dap1 defines the pew2 locus. The mutants at pew1 contain normal levels of immunochemically detectable apoprotein of the phytochrome that is relatively abundant in etiolated seedlings, but are deficient in spectrophotometrically detectable phytochrome, whether seedlings are grown in darkness or light. Moreover, biliverdin, a precursor of the phytochrome chromophore, restores light-regulated responses in pew1 mutants and increases their level of photoreversible phytochrome when grown in darkness. These results indicate that the pew1 locus may be involved in chromophore biosynthesis. The mutant at the pew2 locus displays no photoreversible phytochrome in etiolated seedlings, but does contain normal levels of photoreversible phytochrome when grown in the light. Biliverdin had little effect on light-regulated responses in this mutant. In addition, biliverdin did not alter the level of phytochrome in etiolated seedlings. These observations lead us to propose that this mutant could be affected in the phyA gene itself. We have also obtained the homozygous double mutant at the pew1 and pew2 loci. This double mutant is lethal at an early stage of development, consistent with a critical role for phytochrome in early development of higher plants.

Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating.

The present study examined whether protein kinase C phosphorylated a ciliary protein and whether this phosphorylation event was temporally correlated with a decrease in ciliary beat frequency. Activation of protein kinase C decreased ciliary beat frequency of sheep tracheal epithelium, an effect fully blockable by pretreatment of the tissue pieces with H-7, a protein kinase inhibitor. Using cilia removed from these epithelial surfaces and incubated in solutions containing stimulators of protein kinase C along with [gamma-32P]ATP or [gamma-35S]ATP, a single protein target of ciliary protein kinase C activity was identified. The protein is a polypeptide of molecular mass 37 kDa (p37) as estimated by SDS-polyacrylamide gel electrophoresis. Protein kinase C dependency of p37 phosphorylation was proven by showing that Calphostin C, a specific protein kinase C inhibitor, blocked label incorporation into p37 completely, and by demonstrating that purified protein kinase C phosphorylated p37. Inhibitors of cAMP-dependent kinase and calcium/calmodulin-dependent kinase did not change the phosphorylation of p37 in the presence of protein kinase C activators. p37 was recovered in a Triton X-100-extractable fraction of this ciliary preparation, suggesting that p37 is membrane associated. This hypothesis was further supported by the fact that p37 was present in a pellet representing reconstituted membranes. Thin-layer electrophoresis revealed that p37 was phosphorylated on serine and tyrosine residues, suggesting that the activation of protein kinase C also stimulated tyrosine kinase activity. p37 did not precipitate with annexin I or II antibodies. These results show that sheep tracheal cilia contain protein kinase C activity and that activated protein kinase C phosphorylates a membrane-associated ovine ciliary target, an effect temporally related to a protein kinase C-mediated decrease in ciliary beat frequency.

Cyclic AMP-dependent phosphorylation of a 26 kD axonemal protein in ovine cilia isolated from small tissue pieces.

To study cyclic adenosine monophosphate (cAMP)-dependent phosphorylation events in ovine cilia in vitro, we adapted published axonemal isolation methods to obtain pure mammalian axonemal proteins from small ovine tracheal mucosa pieces with a surface area of only 1 cm2. The isolated axonemes could be reactivated in vitro upon ATP addition, thereby attesting to their functional integrity. The axonemal protein yield from these small mucosa pieces was high enough to allow protein concentration measurements of each sample and axonemal polypeptide analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). cAMP is known to increase ciliary beat frequency, possibly through a phosphorylation event in the axoneme. To study cAMP-dependent phosphorylation events in ovine tracheal cilia, these axonemal preparations were exposed to [gamma-32P]ATP under conditions that stimulated or inhibited kinase activity. Analysis of axonemal polypeptides by SDS-PAGE and subsequent autoradiography showed that an axonemal protein with a M(r) of 26 kD is the only polypeptide consistently phosphorylated in a cAMP-dependent manner. The phosphorylation of this protein could be diminished by a highly specific inhibitor of cAMP-dependent protein kinase, KT-5720. The addition of calcium did not affect label incorporation into this protein during cAMP treatment. In the presence of cAMP and calcium, inhibitors of protein kinase C and calcium/calmodulin-dependent kinase did not change the level of phosphorylation of the 26 kD protein. We conclude that cAMP treatment of isolated mammalian cilia results in the phosphorylation of a single protein with a M(r) of 26 kD (p26).(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal and light regulation of the expression of three phytochromes in germinating seeds and young seedlings of Avena sativa L.

An oat (Avena sativa L.) plant contains at least three phytochromes, which have monomeric masses of 125, 124, and 123 kilodaltons (kDa) (Wang et al., 1991, Planta 184, 96-104). The 124-kDa phytochrome is most abundant in dark-grown seedlings, while the other two phytochromes predominate in light-grown seedlings. Using three monoclonal antibodies, each specific to one of the three phytochromes, we have monitored by immunoblot assay the expression of these three phytochromes in the 5 d following onset of imbibition of seeds. On a per-organism basis, each of these three phytochromes increased in abundance for the first 3 d in the light, or for the first 4 d in darkness, after which they each began to decrease in quantity. When 3-d-old dark-grown seedlings were transferred to the light, the abundance of each of these three phytochromes decreased both in absolute amount and relative to the phytochrome levels in control seedlings kept in darkness. In contrast, when 3-d-old light-grown seedlings were transferred to darkness, the abundance of the 124-kDa and 125-kDa phytochromes increased while that of 123-kDa phytochrome remained unchanged. In each case, the level of phytochrome was greater than that of control seedlings maintained in the light. Thus, in addition to temporal regulation, all three phytochromes exhibit photoregulated expression at the protein level, although the magnitude of this photoregulation varies substantially.

Spatial distribution of three phytochromes in dark- and light-grown Avena sativa L.

We have addressed two issues regarding the spatial distribution of three phytochromes in 3-d-old oat (Avena sativa L.) seedlings. Three monoclonal antibodies, GO-4, GO-7 and Oat-22, were used as probes. Each antibody detects only one of the phytochromes. The first issue is whether any of the phytochromes might be membrane-bound. To address this issue the abundance of each phytochrome in extracts prepared with either a detergent-free or a detergent-containing buffer was compared by immunoblot assay. The detergent-free buffer was formulated to extract only soluble protein, while the detergent-containing buffer was intended to extract both soluble and membrane proteins. None of the data indicate that any of these three phytochromes is membrane-bound in either a dark- or a light-grown seedling. The second issue is whether these three phytochromes are distributed differentially in 3-d-old dark- and light-grown seedlings. When seedlings were dissected into shoots, scutellums, and roots, all three phytochromes were detected in all three fractions from both dark- and light-grown seedlings. Each of the three phytochromes was most abundant in the shoot and least abundant in the root, except that in light-grown seedlings type I, etiolated-tissue phytochrome was more abundant in the root than in either the shoot or the scutellum. When the equivalent fractions dissected from different seedlings were compared, those dissected from dark-grown seedlings contained a higher quantity of each of the three phytochromes than did those dissected from light-grown seedlings, except that green-tissue, type II phytochromes did not differ significantly in the roots. At this level of resolution, no evidence was obtained to indicate a substantive difference among the three phytochromes in their spatial distribution.