Glucocorticoid receptor polymorphisms and haplotypes associated with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a significant public health problem of unknown etiology, the pathophysiology has not been elucidated, and there are no characteristic physical signs or laboratory abnormalities. Some studies have indicated an association of CFS with deregulation of immune functions and hypothalamic-pituitary-adrenal (HPA) axis activity. In this study, we examined the association of sequence variations in the glucocorticoid receptor gene (NR3C1) with CFS because NR3C1 is a major effector of the HPA axis. There were 137 study participants (40 with CFS, 55 with insufficient symptoms or fatigue, termed as ISF, and 42 non-fatigued controls) who were clinically evaluated and identified from the general population of Wichita, KS. Nine single nucleotide polymorphisms (SNPs) in NR3C1 were tested for association of polymorphisms and haplotypes with CFS. We observed an association of multiple SNPs with chronic fatigue compared to non-fatigued (NF) subjects (P < 0.05) and found similar associations with quantitative assessments of functional impairment (by the SF-36), with fatigue (by the Multidimensional Fatigue Inventory) and with symptoms (assessed by the Centers for Disease Control Symptom Inventory). Subjects homozygous for the major allele of all associated SNPs were at increased risk for CFS with odds ratios ranging from 2.61 (CI 1.05-6.45) to 3.00 (CI 1.12-8.05). Five SNPs, covering a region of approximately 80 kb, demonstrated high linkage disequilibrium (LD) in CFS, but LD gradually declined in ISF to NF subjects. Furthermore, haplotype analysis of the region in LD identified two associated haplotypes with opposite alleles: one protective and the other conferring risk of CFS. These results demonstrate NR3C1 as a potential mediator of chronic fatigue, and implicate variations in the 5' region of NR3C1 as a possible mechanism through which the alterations in HPA axis regulation and behavioural characteristics of CFS may manifest.

Improved detection of viral RNA isolated from liquid-based cytology samples.

BACKGROUND: Molecular diagnosis requires the ability to obtain high-quality nucleic acids that are representative of the disease state. We evaluated the recovery and detection of limiting amounts of viral oncogenic RNA from cells fixed in liquid-based cytology media. METHODS AND RESULTS: Serial dilutions of a human papillomavirus (HPV)-positive cell line fixed in a liquid media was used as a model system. Total nucleic acid (TNA) extraction produced RNA with clearly visible ribosomal bands even after one year of storage. These TNA extracts, treated with DNase-I, were used in an RT-PCR assay for HPV-16 E6-E7 oncogenic transcripts. With chemiluminscent Southern blot detection, samples with one HPV-positive cell in 30,000 were consistently detected. CONCLUSION: PreservCyt-fixed cells can yield RNA suitable for molecular assays even after one year of storage.

Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.

We evaluated real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA arrays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavirus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than twofold difference, and the other 144 genes as expressed uniquely or with more than twofold difference between the two subclones. Real-time RT-PCR used LightCycler-based SYBR Green I dye detection and melting curve analysis to validate the relative change in gene expression. Real-time RT-PCR confirmed the change in expression of 17 of 24 (71%) genes identified by high-density filter arrays. Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR. This data suggests that (i) both hybridization intensity and the level of differential expression determine the likelihood of validating high-density filter array results and (ii) genes identified by DNA arrays with a two- to fourfold difference in expression cannot be eliminated as false nor be accepted as true without validation. Real-time RT-PCR based on LightCycler technology is well-suited to validate DNA array results because it is quantitative, rapid, and requires 1000-fold less RNA than conventional assays.

Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies.

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using "hot-start" PCR and empirically determined annealing and signal acquisition temperatures for each gene-specific primer. Relative expression levels were quantified by constructing a standard curve using cDNA dilutions of a highly expressed gene. Using this approach, real-time PCR validated 17 of 21 (71%) genes identified by DNA arrays, and all but 1 of 13 (91%) genes identified by differential display PCR (DD-PCR). Validation of differentially expressed genes detected by array analysis was related to hybridization intensity. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two to fourfold difference in expression cannot be accepted as true or false without validation. Validation of differentially expressed genes detected by DD-PCR was not affected by band intensities. Regardless of the gene expression profiling technology (microarrays, DD-PCR, serial analysis of gene expression and subtraction hybridization), once the sequence of gene of interest is known, the real-time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than conventional assays.

Alternative transcript initiation and novel post-transcriptional processing of a leucine-rich repeat receptor-like protein kinase gene that responds to short-day photoperiodic floral induction in morning glory (Ipomoea nil).

A gene (inrpk1) encoding a putative receptor-like protein kinase was isolated from the Japanese morning glory, Ipo-moea (Pharbitis) nil Roth. cv. Violet. The receptor-like portion of the largest derived polypeptide contains 26 direct leucine-rich repeats (LRRs) in a single block, and the catalytic portion has all the conserved amino acid residues characteristic of Ser/Thr protein kinases. RNA blot analysis detected multiple transcripts in cotyledons. The largest (4.4 kb) transcript encodes the predicted full length polypeptide (INRPK1), whereas a 1.6 kb transcript apparently originates from a secondary transcription initiation site within the gene and potentially encodes a protein kinase identical to INRPK1 but lacking most of the LRRs. Two transcripts (ca. 2.7 and 2.6 kb) are created by alternative 3'-splicing of a large (ca. 1.4-1.5 kb) cryptic intron in the LRR region, creating one transcript (2.6 kb) potentially encoding a small, secretable polypeptide. The larger transcript encoding a polypeptide identical to INRPK1, but lacking 21 LRRs, predominates in vegetative roots. Competitive PCR indicates that inrpk1 mRNA increases 20-fold in cotyledons in response to a previously given single floral-inducing short-day (SD). No differences of this magnitude were detected in any other organs examined from plants similarly treated. This pattern of expression and differential processing suggests a role for inrpk1 in some aspect of SD photoperiodic-induced flowering in morning glory.

Cloning and expression of Shaker alpha- and beta-subunits during inner ear development.

Sensory cells of the chicken cochlea exhibit different ion channels relative to their position along the epithelium. One of these channels conducts an A-type potassium current which is found primarily in 'short' hair cells. Here, we report the first full length cloning and developmental expression of Shaker genes from this endorgan. Clones were obtained by screening a chicken (Gallus gallus) cochlea cDNA library, using probes made from RHK1 (i.e., Kvalpha1.4) cDNA, a Shaker homologue isolated from rat heart, and hKvbeta1.2 cDNA, a beta homologue isolated from human heart. Sequence analysis revealed a chick homologue of Kvalpha1.4, with a deduced amino acid similarity of 76-79% to mammalian Kvalpha1.4, and a chick homologue of Kvbeta1.1, with a similarity of 95% to mammalian Kvbeta1.1. In addition, we isolated a variant of cKvalpha1. 4 (cKvalpha1.4(m)) that differs in its untranslated regions and shows complete similarity in its coding region, except for the deletion of a single nucleotide. During development of the inner ear, reverse transcription-polymerase chain reaction (RT-PCR) studies show that the beta-subunit is expressed as early as embryonic day 3, whereas alpha- and beta-subunits are coexpressed on embryonic days 7 to 10, 14, and in adult.

Chemiluminescent analysis of gene expression on high-density filter arrays.

We have optimized conditions for the chemiluminescent analysis of gene expression using high-density filter arrays (HDFAs). High sensitivity and specificity were achieved by optimizing cDNA probe synthesis, hybridization, and detection parameters. The chemiluminescent expression profile reflected expected differences in the transcripts isolated from different sources (placenta and keratinocytes). We estimated the detection limit for low-abundance message to be 1-15 transcripts per cell, a sensitivity rivaling that reported for microarray formats and exceeding that reported for autoradiographic HDFAs. The method allows for short exposure times and reuse of probe. It should be equally applicable to techniques such as differential screening of cDNA libraries and differential display PCR.

Flower-bud formation in explants of photoperiodic and day-neutral Nicotiana biotypes and its bearing on the regulation of flower formation.

The capacity to form flower buds in thin-layer explants was studied in flowering plants of several species, cultivars, and lines of Nicotiana differing in their response to photoperiod. This capacity was found in all biotypes examined and could extend into sepals and corolla. It varied greatly, depending on genotype, source tissue and its developmental stage, and composition of the culture medium, particularly the levels of glucose, auxin, and cytokinin. It was greatest in the two day-neutral plants examined, Samsun tobacco and Nicotiana rustica, where it extended from the inflorescence region down the vegetative stem, in a basipetally decreasing gradient; it was least in the two qualitative photoperiodic plants studied, the long-day plant Nicotiana silvestris and the short-day plant Maryland Mammoth tobacco, the quantitative long-day plant Nicotiana alata and the quantitative short-day plant Nicotiana otophora line 38-G-81, where it was limited to the pedicels (and, in some cases, the sepals). Regardless of the photoperiodic response of the source plants, the response was the same in explants cultured under long and short days. The finding that capacity to form flower buds in explants is present in all Nicotiana biotypes studied supports the idea that it is regulated by the same mechanism(s), regardless of the plant's photoperiodic character. However, the source plants were all in the flowering stage, and no flower-bud formation can be obtained in explants from strictly vegetative Nicotiana plants. Hence, flower formation in the explants is not identical with de novo flower formation in a hitherto vegetative plant: it is rather the expression of a floral state already established in the plant, although it can vary widely in extent and spatial distribution. Culture conditions that permit flower-bud formation in an explant are conditions that maintain the floral state and encourage its expression; conditions under which no flower buds are formed reduce this state and/or prevent its expression.

Isolation and characterization of cDNA clones for NADP-malic enzyme from leaves of Flaveria: transcript abundance distinguishes C3, C3-C4 and C4 photosynthetic types.

To study the control of enhanced synthesis of enzymes associated with C4 photosynthesis relative to non-C4 plants, we investigated the expression of NADP-malic enzyme (NADP-ME) in different photosynthetic types of Flaveria. Complementary DNA clones encoding NADP-ME were constructed using poly(A)+ RNA from leaves of Flaveria trinervia (C4) and F. linearis (C3-C4) and identified by homology to a cDNA clone (500 bp) encoding NADP-ME from maize (Zea mays L. [39]). The sequence of one clone from each species was determined. The Flaveria clones were 90% homologous over a 564 nucleotide region encoding the carboxy terminal end of the derived polypeptide; sequence similarity to the maize transcript in this region was 71%. Both Flaveria clones detected a 2/3 kb transcript by hybridization to poly(A)+ RNA from expanding leaves of F. trinervia, F. linearis and F. pringlei (C3). The level of transcripts paralleled previously observed NADP-ME activity and abundance differences determined in these species, suggesting that control of the expression of NADP-ME in different photosynthetic types is predominantly at the transcriptional/post-transcriptional level. Southern analysis of genomic DNAs from F. trinervia, F. linearis and F. pringlei indicated a low copy number for this gene in all three species.

Comparison of de-novo flower-bud formation in a photoperiodic and a day-neutral tobacco.

Regeneration of flower buds in thin tissue layers from pedicels of photoinduced short-day (SD) tobacco, Nicotiana tabacum L. cv. Maryland Mammoth, is described. Up to seven flower buds per explant were obtained in a medium containing Murashige and Skoog's macro- and microclements, 100 mg/l myoinositol, 0.1 mg/l thiamine-HCl, 6% glucose, 5 µM N(6)-benzylaminopurine, and 0.5 µM α-naphthaleneacetic acid. Usually some vegetative buds were also formed in the pedicel thin tissue layers. Thin tissue layers from other positions in the induced SD tobacco regenerated vegetative buds only. A comparative study with a day-neutral (DN) tobacco, Samsun, showed that the capacity to form de-novo flower buds was more localized and less strongly determined in photoperiodic than in the DN tobacco. The differences between the photoperiodic and DN tobaccos in flower-bud regeneration capacity are thus quantitative and not qualitative. The basis for this quantitative difference is not known, but may depend on factors controlling production of floral stimulus (florigen) and competency of cells to respond to florigen, and-or stability of the determined state to form flower buds in vitro.