A DNA methylation biomarker of alcohol consumption.

The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42-76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90-0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted P<1 × 10-7. Analysis of the monocyte-derived DNA (n=1251) identified 62 alcohol-related CpGs at P<1 × 10-7. In whole-blood samples of people of European ancestry, we detected differential methylation in two neurotransmitter receptor genes, the γ-Aminobutyric acid-A receptor delta and γ-aminobutyric acid B receptor subunit 1; their differential methylation was associated with expression levels of a number of genes involved in immune function. In conclusion, we have identified a robust alcohol-related DNA methylation signature and shown the potential utility of DNA methylation as a clinically useful diagnostic test to detect current heavy alcohol consumption.

Association of heat shock proteins with all-cause mortality.

Experimental mild heat shock is widely known as an intervention that results in extended longevity in various models along the evolutionary lineage. Heat shock proteins (HSPs) are highly upregulated immediately after a heat shock. The elevation in HSP levels was shown to inhibit stress-mediated cell death, and recent experiments indicate a highly versatile role for these proteins as inhibitors of programmed cell death. In this study, we examined common genetic variations in 31 genes encoding all members of the HSP70, small HSP, and heat shock factor (HSF) families for their association with all-cause mortality. Our discovery cohort was the Rotterdam study (RS1) containing 5,974 participants aged 55 years and older (3,174 deaths). We assessed 4,430 single nucleotide polymorphisms (SNPs) using the HumanHap550K Genotyping BeadChip from Illumina. After adjusting for multiple testing by permutation analysis, three SNPs showed evidence for association with all-cause mortality in RS1. These findings were followed in eight independent population-based cohorts, leading to a total of 25,007 participants (8,444 deaths). In the replication phase, only HSF2 (rs1416733) remained significantly associated with all-cause mortality. Rs1416733 is a known cis-eQTL for HSF2. Our findings suggest a role of HSF2 in all-cause mortality.

Genome-wide meta-analyses of smoking behaviors in African Americans.

The identification and exploration of genetic loci that influence smoking behaviors have been conducted primarily in populations of the European ancestry. Here we report results of the first genome-wide association study meta-analysis of smoking behavior in African Americans in the Study of Tobacco in Minority Populations Genetics Consortium (n = 32,389). We identified one non-coding single-nucleotide polymorphism (SNP; rs2036527[A]) on chromosome 15q25.1 associated with smoking quantity (cigarettes per day), which exceeded genome-wide significance (ß = 0.040, s.e. = 0.007, P = 1.84 × 10(-8)). This variant is present in the 5'-distal enhancer region of the CHRNA5 gene and defines the primary index signal reported in studies of the European ancestry. No other SNP reached genome-wide significance for smoking initiation (SI, ever vs never smoking), age of SI, or smoking cessation (SC, former vs current smoking). Informative associations that approached genome-wide significance included three modestly correlated variants, at 15q25.1 within PSMA4, CHRNA5 and CHRNA3 for smoking quantity, which are associated with a second signal previously reported in studies in European ancestry populations, and a signal represented by three SNPs in the SPOCK2 gene on chr10q22.1. The association at 15q25.1 confirms this region as an important susceptibility locus for smoking quantity in men and women of African ancestry. Larger studies will be needed to validate the suggestive loci that did not reach genome-wide significance and further elucidate the contribution of genetic variation to disparities in cigarette consumption, SC and smoking-attributable disease between African Americans and European Americans.

Association analysis of SNPs in the IL4R locus with type I diabetes.

The Type I Diabetes Genetics Consortium (T1DGC) has collected thousands of multiplex and simplex families with type I diabetes (T1D) with the goal of identifying genes involved in T1D susceptibility. These families have all been genotyped for the HLA class I and class II loci and a subset of samples has been typed for an major histocompatibility complex (MHC) single-nucleotide polymorphism (SNP) panel. In addition, the T1DGC has genotyped SNPs in candidate genes to evaluate earlier reported T1D associations. Individual SNPs and SNP haplotypes in IL4R, which encodes the alpha-chain of the IL4 and IL13 receptors, have been associated with T1D in some reports, but not in others. In this study, 38 SNPs in IL4R were genotyped using the Sequenom iPLEX Gold MassARRAY technology in 2042 multiplex families from nine cohorts. Association analyses (transmission-disequilibrium test and parental-disequilibrium test) were performed on individual SNPs and on three-SNP haplotypes. Analyses were also stratified on the high-risk HLA DR3/DR4-DQB1*0302 genotype. A modest T1D association in HBDI families (n=282) was confirmed in this larger collection of HBDI families (n=424). The variant alleles at the non-synonymous SNPs (rs1805011 (E400A), rs1805012 (C431R), and rs1801275 (Q576R)), which are in strong linkage disequilibrium, were negatively associated with T1D risk. These SNPs were more associated with T1D among non-DR3/DR4-DQB1*0302 genotypes than DR3/DR4-DQB1*0302 genotypes. This association was stronger, both in terms of odds ratio and P-values, than the initial report of the smaller collection of HBDI families. However, the IL4R SNPs and the three-SNP haplotype containing the variant alleles were not associated with T1D in the total data. Thus, in the overall families, these results do not show evidence for an association of SNPs in IL4R with T1D.

Overview of the Rapid Response data.

The Type I Diabetes Genetics Consortium (T1DGC) Rapid Response Workshop was established to evaluate published candidate gene associations in a large collection of affected sib-pair (ASP) families. We report on our quality control (QC) and preliminary family-based association analyses. A random sample of blind duplicates was analyzed for QC. Quality checks, including examination of plate-panel yield, marker yield, Hardy-Weinberg equilibrium, mismatch error rate, Mendelian error rate, and allele distribution across plates, were performed. Genotypes from 2324 families within nine cohorts were obtained from a panel of 21 candidate genes, including 384 single-nucleotide polymorphisms on two genotyping platforms performed at the Broad Institute Center for Genotyping and Analysis (Cambridge, MA, USA). The T1DGC Rapid Response project, following rigorous QC procedures, resulted in a 2297 family, 9688 genotyped individual database on a single-candidate gene panel. The available data include 9005 individuals with genotype data from both platforms and 683 individuals genotyped (276 in Illumina; 407 in Sequenom) on only one platform.

Overview of the MHC fine mapping data.

AIM: The aim of this study was to perform quality control (QC) and initial family-based association analyses on the major histocompatibility complex (MHC) single nucleotide polymorphism (SNP) and microsatellite marker data for the MHC Fine Mapping Workshop through the Type 1 Diabetes Genetics Consortium (T1DGC). METHODS: A random sample of blind duplicates was sent for analysis of QC. DNA samples collected from participants were shipped to the genotyping laboratory from several T1DGC DNA Repository sites. Quality checks including examination of plate-panel yield, marker yield, Hardy-Weinberg equilibrium, mismatch error rate, Mendelian error rate and allele distribution across plates were performed. RESULTS: Genotypes from 2325 families within nine cohorts were obtained and subjected to QC procedures. The MHC project consisted of three marker panels - two 1536 SNP sets (Illumina Golden Gate platform performed at the Wellcome Trust Sanger Institute, Cambridge, UK) and one 66 microsatellite marker panel (performed at deCODE). In the raw SNP data, the overall concordance rate was 99.1% (+/-0.02). CONCLUSIONS: The T1DGC MHC Fine Mapping project resulted in a 2300 family, 9992 genotyped individuals database comprising of two 1536 SNP panels and a 66 microsatellite panel to densely cover the 4 Mb MHC core region for use in statistical genetic analyses.

Hormone replacement therapy is associated with increased C-reactive protein in women with Type 2 diabetes in the Diabetes Heart Study.

AIMS: Increased levels of inflammatory biomarkers, especially C-reactive protein (CRP), are associated with increased risk for cardiovascular disease (CVD) events, such as myocardial infarction, stroke, peripheral vascular disease, and sudden cardiac death. Medical interventions that increase CRP levels, such as hormone replacement therapy (HRT) in post-menopausal women, are under increasing scrutiny. The effect of HRT on CRP levels in women with Type 2 diabetes (T2DM) is not well documented, and conflicting conclusions have been reported. The aim of this study was to determine the influence of HRT on women with diabetes in a large cross-sectional study. METHODS: Three hundred and twenty-seven post-menopausal women with T2DM from the Diabetes Heart Study participated. Current use of HRT was determined and serum CRP levels were measured using a high-sensitivity ELISA kit. Generalized estimating equation methods were used to assess the relationship of multiple clinical and lifestyle (e.g. smoking) measures on CRP levels including differences between women taking HRT (HRT+) and not taking HRT (HRT-). RESULTS: Overall serum CRP levels were strongly associated with body mass index (P < 0.0001) and age (P < 0.0001). Of the women, 243 were not using HRT and 84 were using HRT. HRT+ and HRT- women did not differ significantly in measures of clinical traits, with the exception of higher mean low-density lipoprotein cholesterol in HRT- women (P = 0.004). In all models tested, HRT+ women had significantly higher circulating CRP levels, with P-values ranging from 0.0045 to 0.010. CONCLUSIONS: In this study of serum CRP concentration as a function of HRT in women with Type 2 diabetes, there was consistent evidence for increased circulating CRP levels in women receiving oestrogen-containing HRT. Whether HRT-induced increases in CRP can account for the adverse cardiovascular effects of HRT remains to be established; however, based on these data, there is little reason to believe that diabetic women would be spared from such an effect.

Type 2 diabetes is not independently associated with spinal trabecular volumetric bone mineral density measured by QCT in the Diabetes Heart Study.

The purpose of this study was to investigate the association between type 2 diabetes mellitus (DM2) and trabecular volumetric bone mineral density (vBMD) of the thoracic and lumbar spine measured by quantitative computed tomography (QCT) in 483 female (410 with DM2) and 398 male (365 with DM2) adults (age 36-86 years, BMI 16-58, 88% with DM2) in the Diabetes Heart Study. After accounting for familial correlation using generalized estimating equations (GEE), lumbar spine vBMD was positively associated with BMI (r = 0.24, P < 0.0001) and inversely associated with age (r = -0.51, P < 0.0001). In women, age-adjusted thoracic spinal vBMD (mg/ml, mean +/- SE) was higher in diabetics (147.6 +/- 2.3) compared to unaffected individuals (138.6 +/- 3.4) (P = 0.02), with age-adjusted lumbar spinal vBMD showing a similar but non-significant trend (132.9 +/- 2.1 in diabetics vs. 127.2 +/- 3.6 in unaffected individuals, P = 0.15). In contrast, in men, age-adjusted lumbar and thoracic vBMD were not different between diabetics and unaffected controls (lumbar vBMD = 125.0 +/- 1.8 in diabetics and 125.8 +/- 5.6 in unaffected individuals, P = 0.89; thoracic vBMD = 137.4 +/- 2.1 in diabetics vs. 134.2 +/- 5.5 in controls, P = 0.56). After multivariate analysis adjusting for age, sex, race, BMI, physical activity, dietary intake, smoking, and alcohol use, interaction between diabetes status and trabecular vBMD of the spine was no longer observed. In women only, age-adjusted areal BMD (determined by dual X-ray absorptiometry (DXA)) of the spine and hip were significantly higher in diabetics than non-diabetic (all P < 0.05), although the differences disappeared after additional adjustment for BMI. These data suggest that areal BMD measured by DXA and trabecular volumetric BMD measured by QCT are not associated with type 2 diabetes independently from BMI.

Heritability of spinal trabecular volumetric bone mineral density measured by QCT in the Diabetes Heart Study.

The heritability of trabecular volumetric bone mineral density (BMD) determined by quantitative computed tomography (QCT) has not yet been reported. The purpose of this study was to investigate the heritability of BMD as determined by QCT and DXA in 124 women and 120 men (age 39-83 years, BMI 17-75, 84% type 2 diabetics) from 101 families (232 sibling pairs) in the Diabetes Heart Study. Volumetric BMD had a heritability (h2) estimate of 0.73 (SE = 0.15, P < 0.0001) at the lumbar spine and 0.71 (SE = 0.15, P < 0.0001) at the thoracic spine. Areal BMD heritability estimates were 0.56 for PA spine, 0.43 for total hip, 0.43 for femoral neck, 0.45 for distal radius, 0.42 for mid-radius, and 0.52 for whole body (all P < 0.01). After accounting for familial correlation using generalized estimating equations, volumetric BMD was inversely associated with age (r = -0.52, P < 0.0001) and duration of diabetes (r = -0.24, P < 0.01) and positively associated with body weight (r = 0.25, P < 0.01). In multivariate analysis, adjustment for age, sex, and race lowered the h2 estimates for volumetric BMD at the lumbar (h2 = 0.41, P < 0.01) and thoracic (h2 = 0.48, P < 0.001) spine, increased the h2 estimate for areal BMD at the mid radius (h2 = 0.58, P < 0.0001), and had little effect on the h2 estimate for areal BMD at other sites (h2 = 0.41-0.55, all P < 0.01). Additional adjustment for BMI, duration of diabetes, and physical activity had little effect on the h2 estimates for volumetric BMD or areal BMD except at the hip where they were lowered (h2 = 0.31-0.33, all P < 0.05). These data suggest that, like areal BMD, volumetric BMD is highly heritable and may be used in designing linkage studies to locate genes governing bone metabolism.

Development of real-time RT-PCR for the detection of avian influenza virus.

A real-time reverse transcriptase/polymerase chain reaction (RRT-PCR) assay was developed using hydrolysis probes for the detection of avian influenza virus (AIV) and the H5 and H7 subtypes. The AIV specific primers and probes were directed to regions of the AIV matrix gene that are conserved among most type A influenza viruses. The H5 and H7 primers and probes are directed to H5 and H7 hemagglutinin gene regions that are conserved among North American avian influenza viruses. The sensitivity and specificity of this RRT-PCR assay was compared to virus isolation (VI) in chicken embryos with 1550 clinical swab samples from 109 live-bird markets (LBMs) in New York and New Jersey. RRT-PCR detected influenza in samples from 61 of 65 (93.8%) of the LBMs that were the sources of VI positive samples. Of the 58 markets that were positive for H7 influenza by hemagglutination inhibition assay, RRT-PCR detected H7 influenza in 56 markets (96.5%). Too few H5 positive samples were obtained to validate the H5 RRT-PCR assay in this study. Although RRT-PCR was less sensitive than VI on an individual sample basis, this study demonstrated that the AIV and H7 RRT-PCR assays are good tools for the rapid screening of flocks and LBMs.

Adiponectin as a novel determinant of bone mineral density and visceral fat.

Growing evidence suggests that positive associations between fat mass (FM) and bone mineral density (BMD) are mediated by not only biomechanical but also biochemical factors. Adiponectin is a novel adipocyte-derived hormone that regulates energy homeostasis and has anti-inflammatory and anti-atherogenic effects. Unlike other adipokines such as leptin, adiponectin levels decrease in obesity and type 2 diabetes. The purpose of our study was to investigate associations of serum adiponectin with BMD (DXA and QCT), FM (DXA and QCT), and serum leptin and soluble leptin receptor levels in 38 women and 42 men (age 39-81, BMI 17-55, 86% with type 2 diabetes). After adjusting for age, gender, race, smoking, and diabetes status, serum adiponectin was inversely associated with areal BMD (r = -0.20 to -0.3, all P < 0.01), volumetric BMD (r = -0.35 to -0.44, all P < 0.01), and visceral fat volume (r = -0.30, P < 0.01). These associations remained significant after adjusting for whole body fat mass. The associations of adiponectin with subcutaneous fat volume, whole body FM, and serum leptin level were not significant (all P > 0.1). These data suggest that adiponectin may play a role in the protective effects of visceral fat on BMD.

Atmospheric fate and transport of dioxins: local impacts.

We conducted model simulations of the atmospheric fate and transport of PCDD/F to assess the fraction of emitted PCDD/F that would deposit within 100 km from the source. We considered eight major categories of PCDD/F emission sources and six different locations, to cover a wide range of source characteristics, PCDD/F congener profiles and particle size distributions, meteorological conditions and terrain configurations. These results suggest that for sources that have tall stacks and/or high plume rise (e.g., copper smelters, cement kilns, sinter plants), only a small fraction of PCDD/F emissions is deposited locally (typically, less than 10% within 100 km). Other source categories such as municipal solid waste incinerators, medical waste incinerators and diesel trucks lead to a greater fraction of PCDD/F being deposited locally; nevertheless, the majority of their PCDD/F emissions tends to be transported beyond 100 km. Although local impacts may need to be addressed for these latter source categories, it appears that the long-range potential impacts of PCDD/F need to be addressed for all source categories. Sensitivity studies were conducted to investigate the effect of various key model inputs on simulation results. These studies suggest that an advanced atmospheric dispersion model should be used for cases where PCDD/F emissions may present some local concerns because the results are very sensitive to the treatment of dispersion. Also, it is essential to obtain accurate characterizations of the particle size distribution of particulate PCDD/F because the dry deposition flux is very sensitive to the particle size distribution.

Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.

Phylogeography of the tailed frog (Ascaphus truei): implications for the biogeography of the Pacific Northwest.

Tailed frogs are distributed in high-gradient streams within the disjunct mesic forests of the Pacific Northwest and represent the basal lineage of the anurans. We sequenced 1,530 nucleotides of the mitochondrial cytochrome b and NADH dehydrogenase subunit two genes from 23 populations and used parsimony, maximum-likelihood, and nested-clade analyses to estimate relationships among populations and infer evolutionary processes. We found two divergent haplotype clades corresponding with inland Rocky Mountain populations and coastal populations and separated by up to 0.133 substitutions per site. Within the coastal assemblage, haplotypes formed clades by mountain range with 0.010-0.024 substitutions per site divergence among populations. Inland haplotypes exhibited minimal genetic structure, with the exception of 0.021 substitutions per site distance between populations from the East Fork of the South Fork of the Salmon River and all other inland haplotypes. The magnitude of divergence between inland and coastal populations, as well as the paleobotanical record, suggest isolation of these lineages occurred during the late Miocene to early Pliocene, probably in response to the rise of the Cascade Mountains. Genetic structure within coastal and inland populations is consistent with isolation in refugia during the late Pliocene and early Pleistocene. Closely related inland haplotypes reflect range expansion following glaciation. The depth of divergence between inland and coastal populations supports the persistence of mesic forests within the inland Pacific Northwest throughout the Pleistocene and is congruent with patterns found in several other mesic forest species. Based on mitochondrial divergence and previous allozyme and morphological data, we recommend recognition of inland populations as a distinct species, Ascaphus montanus.

High-density lipoprotein cholesterol and vascular stiffness at baseline in the activity counseling trial.

In a middle-aged patient population, age was associated with stiffer vessels and high-density lipoprotein cholesterol with more elastic vessels. High-density lipoprotein cholesterol may be an indirect indicator of aerobic capacity or of less atherosclerosis, suggesting mechanisms for preserving vascular integrity.

Sensitivity analysis of mercury human exposure.

We present a comprehensive analysis of the sensitivity of mercury (Hg) human exposure to environmental variables using a multimedia model of the fate and transport of Hg in the environment. The results of the analysis show that the Hg dose is most sensitive to the lake pH, the burial rate of Hg adsorbed to sediments, and the chemical speciation of Hg emissions to the atmosphere. The lake pH has a strong non-linear effect on the methylation rate and bioaccumulation of Hg in fish. The burial of sediments is a major pathway for removing Hg from the lake cycling. The speciation of Hg emissions is important because Hg(II) is deposited much more rapidly than Hg(0). These results highlight the importance of key variables that should be investigated through well-designed field programs, so that we can minimize the overall uncertainties associated with the modeling of mercury fate and transport.

The Department of Defense laboratory-based global influenza surveillance system.

Military global influenza surveillance began in 1976 as an Air Force program. In 1997, the Department of Defense (DoD) Global Emerging Infections Surveillance and Response System expanded the program to include all services. Also included were local residents in areas where DoD overseas research activities operated. This new, worldwide DoD surveillance infrastructure provides valuable information and can respond quickly to outbreaks. This was demonstrated during the current influenza season when a suspected outbreak was reported in Panama. In less than 3 weeks, specimens were collected, transported, and cultured, and isolates were subtyped and sent to the Centers for Disease Control and Prevention for further studies. This influenza surveillance initiative combines viral isolation, antigenic characterization, and molecular sequencing with clinical and public health management of information. The information obtained is shared with the Centers for Disease Control and Prevention and the World Health Organization and has contributed to important decisions in influenza vaccine composition.

Phase II trial of adjuvant radiation and intraperitoneal 5-fluorouracil for locally advanced colon cancer: results with 10-year follow-up.

PURPOSE: To determine the toxicity, disease-free survival, and overall survival for patients with Modified Astler-Coller (MAC) B2-3 or C1-3 colon cancer receiving adjuvant radiation and sequential intraperitoneal 5-fluorouracil (5-FU). METHODS AND MATERIALS: From August 1984 to June 1989, 45 patients were accrued to this Phase II trial and received a 21-week course of intraperitoneal 5-FU (20 mg/kg/d x 5) and external beam radiation. The radiation was delivered to the tumor bed and para-aortic lymph nodes in two split-courses of 22.5 Gy, alternating with the first two cycles of chemotherapy. All patients then received 4 additional cycles of intraperitoneal 5-FU. RESULTS: The therapy was well tolerated with 4 patients experiencing Grade 3 peritonitis. Four patients developed small bowel obstruction requiring surgery; in each instance, recurrent tumor was found at the time of laparotomy. The median and overall survivals at 10 years were 9.3 months and 53% respectively. Local failures were infrequent, occurring in only 11% of patients treated. CONCLUSIONS: Sequential intraperitoneal 5-FU and tumor-bed/para-aortic irradiation is tolerable in patients with resected colon cancer. Although the incidence of local and regional relapse appeared to be lower than anticipated, this did not appear to translate into improved survival.

Identification of three genes expressed primarily during development in Physarum polycephalum.

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation.

Sequences within both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation.

Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr from initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.