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Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration.

Adulteration of goat meat with cheaper meat such as pork has been frequently found. Conventional PCR methods to distinguish goat meat from adulteration are qualitative, which easily produce false‐positive results due to contamination but not adulteration. To address this problem, real‐time PCR based on single‐copy housekeeping genes encoding replication protein A1 was developed for goat meat and pork. By calculating the Ct ratio of goat meat/pork, the goat meat content in a suspected adulteration could be deduced by a good linear correlation (R² = 0.9868) in a range from 5% to 80%. Analysis of the simulated samples of goat meat showed high accuracy with recoveries of 104.91% and 105.00% for the goat meat contents of 40% and 60%, respectively, and coefficient of variations were as low as 9.24% and 9.09%. Thus, the developed assay supplied a useful tool for food regulation authorities to achieve quantitative authentication of goat meat.

BACKGROUND Although shorter telomeres have been associated with Alzheimer's disease (AD), it is unclear whether longitudinal change in telomere length is associated with AD progression. OBJECTIVE To investigate the association of telomere length change with AD diagnosis and progression. METHODS In 653 individuals from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort, T/S ratio (telomere versus single copy gene), a proxy of telomere length, was measured for up to five visits per participant (N = 1918 samples post-QC) using quantitative PCR (qPCR). T/S ratio was adjusted for batch effects and DNA storage time. A mixed effects model was used to evaluate association of telomere length with AD diagnostic group and interaction of age and diagnosis. Another mixed effects model was used to compare T/S ratio changes pre- to post-conversion to MCI or AD to telomere change in participants with stable diagnoses. RESULTS Shorter telomeres were associated with older age (Effect Size (ES) = -0.23) and male sex (ES = -0.26). Neither baseline T/S ratio (ES = -0.036) nor T/S ratio change (ES = 0.046) differed significantly between AD diagnostic groups. MCI/AD converters showed greater, but non-significant, telomere shortening compared to non-converters (ES = -0.186). CONCLUSIONS Although AD compared to controls showed small, non-significant effects for baseline T/S ratio and T/S ratio shortening, we did observe a larger, though still non-significant effect for greater telomere shortening in converters compared to non-converters. Although our results do not support telomere shortening as a robust biomarker of AD progression, further investigation in larger samples and for subgroups of participants may be informative.

Objectives Telomeres are structures at the extremity of chromosomes that prevents genomic instability, and its shortening seems to be a hallmark of cellular aging. Past studies have shown contradictory results of telomere length (TL) in major depression, and are a few studies in late‐life depression (LLD). This explores the association between TL as a molecular marker of aging and diagnosis of LLD, the severity of depressive symptoms, and cognitive performance in older adults. Methods/design We included 78 older adults (45 with LLD and 33 nondepressed controls, according to DSM‐V criteria), aged 60–90 years. TL was measured in leukocytes by a quantitative polymerase chain reaction, determining the relative ratio (T/S) between the telomere region copy number (T) and a single copy gene (S), using a relative standard curve. Results TL was significantly shorter in the LLD compared with control participants (p = .039). Comparing groups through the severity of depressive symptoms, we found a negative correlation with the severity of depressive symptoms (Hamilton Depression Rating Scale‐21, r = −0.325, p = .004) and medical burden (r = −0.271, p = .038). There was no significant correlation between TL and cognitive performance (Mattis Dementia Rating Scale, r = 0.152, p = .21). Conclusions We found that older adults with LLD have shorter telomere than healthy controls, especially those with a more severe depressive episode. Our findings suggest that shorter TL can be a marker of the severity of depressive episodes in older adults and indicate that these individuals may be at higher risk of age‐associated adverse outcomes linked to depression., Our study explores and examines the relationship between telomere length (TL) and Late‐Life Depression (LLD), considering the severity of depressive episodes. We found that TL was significantly shorter in the LLD than control participants and was negatively correlated with the severity of depressive symptoms and medical burden. Our findings suggest that shorter TL can be a marker of the severity of depressive episodes in older adults and indicate that these individuals may be at higher risk of age‐associated adverse outcomes linked to depression.

Objective Studies of multiple species have found that adverse early life experiences, including childhood trauma and maternal separation, can result in accelerated telomere shortening. The objective of this study was to determine if premature separation from the mother affected telomere length in domestic kittens (Felis catus). Subjects were 42 orphaned kittens and 10 mother-reared kittens from local animal rescue groups and shelters. DNA was extracted from whole blood collected from kittens at approximately 1 week and 2 months of age. Telomere length was assessed by qPCR (quantitative polymerase chain reaction) from a total of 86 samples and expressed as a ratio of telomere PCR relative to a single copy gene PCR (T/S). Results A generalized linear mixed model found there were no detectable differences in telomere length based on survival (F1, 76.2 = 3.35, p = 0.07), orphan status (F1, 56.5 = 0.44, p = 0.51), time point (F1, 43.5 = 0.19, p = 0.67), or the interaction between orphan status and time (F1, 43.5 = 0.86, p = 0.36). Although in other species telomere shortening is commonly associated with aging, even early in life, we did not find evidence for telomere shortening by two months of age. Our results suggest that the experience of early maternal separation in domestic cats who are subsequently hand-reared by humans does not accelerate telomere shortening compared to mother-reared kittens, at least in the first few months of life.

Background Telomeres are vital for genomic integrity and telomere length has been linked to many adverse health outcomes. Some hazardous air pollutants, or air toxics, increase oxidative stress and inflammation, two possible determinants of shortened telomere length. No studies have examined air toxic-telomere length associations in a non-occupational setting. Methods This study included 731 Sister Study participants (enrolled 2003-2007) who were randomly selected to assess telomere length in baseline blood samples. Multiplex qPCR was used to determine telomere to single copy gene (T/S) ratios. Census tract concentration estimates of 29 air toxics from the 2005 National Air Toxics Assessment were linked to baseline residential addresses. Air toxics were classified into tertile-based categories of the exposure. Multivariable linear regression was used to estimate β coefficients and 95% confidence intervals (CI) in single pollutant models. Multipollutant groups were identified with regression trees. Results The average T/S ratio was 1.24. Benzidine (T3vsT1 β= -0.08; 95% CI: -0.14, -0.01) and 1,4-dioxane (T3vsT1 β= -0.06; 95% CI: -0.13, 0.00) in particular, as well as carbon tetrachloride, chloroprene, ethylene dibromide, and propylene dichloride, were associated with shorter relative telomere length. Benzidine (p=0.02) and 1,4-dioxane (p=0.06) demonstrated some evidence of a monotonic trend. The regression tree identified age, BMI, physical activity, ethylene oxide, acrylonitrile, ethylidene dichloride, propylene dichloride, and styrene in multipollutant groups related to telomere length. Conclusions In this first study of air toxics and telomere length in a non-occupational setting, several air toxics, particularly 1,4-dioxane and benzidine, were associated with shorter relative telomere length.

Bachground The premature ovarian failure (POF) is a reason of infertility that affects about 1–4% of women before age 40. The importance of telomeres' length in different diseases has been explored before. This study examines the association between the relative telomere length and idiopathic POF in a group of Iranian women. Methods The blood genomic DNA was extracted from 40 idiopathic POF patients (case group) and 40 fertile women (control group). The relative telomere length (RTL) was evaluated by quantitative Real-Time PCR using specific telomeric primers. RTL was calculated as T (telomere)/S (single copy gene) ratio and compared between infertile and fertile groups. Results A strong association was considered between telomere size and idiopathic premature ovarian failure. In patients the relative telomere length showed to be significantly longer than those of control group (P Conclusion Our findings demonstrate a possible relationship between telomere lengthening and idiopathic POF. The reason of the observed elevated telomeres' genetic material could be explained by numerous probable mechanisms like reduced ovarian cell division rate, a sudden increase in the estrogen level before menopause and after egg depletion, and/or an autoimmune condition which could change the composition of blood cells and their consequent conversion to the cells with longer telomeres in patients. More experiments with larger population are necessary to confirm the results of present study.

A novel real-time polymerase chain reaction (PCR) method for detecting chickpea was developed. From homologous sequences of 9-cis-epoxycarotenoid dioxygenase gene (NCED) among leguminous species, chickpea’s NCED was cloned, and the Southern-blot analysis showed that NCED is a single copy gene in a haploid genome. Its coding sequences at the 5′ terminus were found unique to chickpea and conserved among various chickpea varieties. Developed real-time PCR method targeting the unique sequences was specific to chickpea and had detection limit of approximately 30 copies per a reaction, and applicable for qualitative detection of chickpea in various forms of food products including dried, powdered, retort-packed, canned, fermented and pasted. Our results showed that the developed method enables identification of chickpea in foods.

Meat adulteration has posed considerable risks to public health. In this study, we developed a novel real-time quantitative PCR method for the detection of some mammalian and poultry species that are used as meat products or meat adulterants. The method was based on the detection of the single-copy nuclear gene myostatin . The specificity, heterogeneity, and copy number of myostatin were evaluated. Additionally, we determined the sensitivity and precision of the method. The results revealed that myostatin had high specificity and low heterogeneity among different mammalian and poultry species. The limit of detection was 5 pg of animal genomic DNA or 0.001% meat ingredient, and the limit of quantification was 10 pg of animal genomic DNA or 0.01% meat ingredient. The quantification results of 12 blind samples showed that the biases between the measured and true values were

Telomeres are repetitive noncoding deoxynucleotide sequences that cap chromosomes to protect DNA. Telomere length (TL) is affected by both genetic and environmental factors, and shortening of telomeres is associated with multiple neuropsychiatric disorders, early life stress, and age-related cognitive dysfunction. Two previous studies associated shorter TL with autism spectrum disorder (ASD). We aimed to replicate this finding, describe TL in unaffected siblings, and explore novel relationships with symptoms and cognitive function in families with ASD. Participants were 212 male children and adolescents ages 1-17 years (86 with ASD, 57 unaffected siblings, and 69 typically developing [TD]) and 64 parents. TL was measured from blood leukocytes with quantitative real-time polymerase chain reaction and results are expressed by relative ratios with a single copy gene. We replicated that children and adolescents with ASD have shorter TL, compared to TD, and show that unaffected siblings have TL in between those of TD and ASD. We present novel associations between TL and sensory symptoms in ASD. Finally, we demonstrate cognitive functions, but not autistic traits, are related to TL in parents of children with ASD. Cognitive function and TL were not related in children and adolescents. As the third replication, our results elicit confidence in the finding that ASD is associated with shorter TL. Our novel sensory investigation suggests that shortened TL may be a biological mechanism of sensory symptoms in ASD. Furthermore, results highlight the need to better understand relationships between cognition, aging, and TL in families with ASD. Autism Res 2020, 13: 1094-1101. © 2020 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Telomeres cap chromosomes to protect DNA. They progressively shorten as people age and are related to health outcomes. We replicated previous findings that children and adolescents with autism spectrum disorder (ASD) have shorter telomeres, compared to typically developing (TD), and show that unaffected siblings have telomere length (TL) in between those of TD and ASD. We find shortened TL is related to more severe sensory symptoms. This may mean families with ASD, especially those with elevated sensory symptoms, are at risk for worse age-related health outcomes.

Exposure to polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) may change leukocyte telomere length (TL) at the end of the DNA sequence. The purpose of this study was to investigate the association between PCBs and OCPs exposure with TL in Tehran adult males. Whole-blood samples were randomly taken from three hundred adult males in population-based cross-section study from October 2016 to November 2017. We studied the serum levels of PCBs, OCPs as well as socio-demographic characteristics of individuals. The quantitative PCR was used to investigate the number of the telomere (T) repeats to the number of a single copy gene. We measured the effect of each PCBs and OCPs congeners on TL using linear regressions adjusted for age, BMI, smoking, and dietary patterns. The median level of the six non-dioxin-likes, five dioxin-likes PCBs three OCPs and TL in the study population were 344.5, 306.0, 45.0 ng/g lipid and 5377.7 ± 573.4 base pairs, respectively. In the adjusted model, the percent difference in the TLs with exposure to Σnon–dioxin-like PCBs, Σdioxin-like PCBs, and OCPs were 1.93 (−0.70 to 5.4), 3.4 (1.8–8.3) and −2.4 (−0.80 to −6.2), respectively. In the fourth quartile compared to the first quartile, the percent difference in the TLs due to Σnon–dioxin-like PCBs, Σdioxin-like PCBs, and OCP exposure were 0.01 (−0.01 to 0.05), 10.3 (2.9–18.1) and −0.20 (−0.10 to −4.5), respectively. Exposures to ndl-PCBs and dl-PCBs (except for PCB28) were related to longer TLs, but OCPs exposure can be related to telomere shortening.

Telomere length (TL) comparisons from different methods are challenging due to differences in laboratory techniques and data configuration. This study aimed to assess the validity of converting the quantitative polymerase chain reaction (qPCR) telomere/single copy gene (T/S) ratio to TL in kilobases (kb). We developed a linear regression equation to predict TL from qPCR T/S using flow cytometry with fluorescence in situ hybridization (flow FISH) TL data from 181 healthy donors (age range = 19&ndash, 53) from the National Marrow Donor Program (NMDP) biorepository. TL measurements by qPCR and flow FISH were modestly correlated (R2 = 0.56, p &lt, 0.0001). In Bland-Altman analyses, individuals with the shortest (&le, 10th percentile) or longest (&ge, 90th) flow FISH TL had an over- or under-estimated qPCR TL (bias = 0.89 and &minus, 0.77 kb, respectively). Comparisons of calculated TL from the NMDP samples and 1810 age- and sex-matched individuals from the National Health and Nutrition Examination Survey showed significant differences (median = 7.1 versus 5.8 kb, respectively, p &lt, 0.0001). Differences in annual TL attrition were also noted (31 versus 13 bp/year, respectively, p = 0.02). Our results demonstrate that TL calculated in kb from qPCR T/S may yield biased estimates for individuals with the shortest or longest TL, those often of high clinical interest. We also showed that calculated TL in kb from qPCR data are not comparable across populations and therefore are not necessarily useful.

The large scale genome datasets that are now available can provide unprecedented insight into the evolution of genes and gene families. Nevertheless, handling and transforming such datasets into the desired format for downstream analyses is often a difficult and time-consuming task for researchers without a background in informatics. Here, we present a simple and fast protocol for data preparation and high quality phylogenetic tree inferences using simple to install cross-platform software applications with rich graphical interfaces. To illustrate its potential, this protocol was used to provide insight into the evolution of GULO gene in animals, a gene that encodes the enzyme responsible for the last step of vitamin C synthesis in this group of organisms. We find that GULO is always a single copy gene in all animal groups with the exception of Echinodermata. Surprisingly, we find potentially functional GULO genes in several Prostotomian groups such as Molluscs, Priapulida and Arachnida. To our knowledge, this is the first time a putative functional GULO gene is reported in Protostomians. All previously reported GULO gene losses were easily identified using the presented protocol.

The objective of this study is to investigate placental telomere shortening in unexplained stillbirths (SBs) as an indication of premature senescence.Placentas were collected from 42 unexplained SB (22 weeks), 43 term and 15 preterm live births, at the Policlinico Hospital of Modena (Italy). DNA extracted from placentae was studied for telomere length by real time PCR. Standard curves were generated for telomere lengths from single copy gene amplifications using a reference DNA. The telomere length for each sample was derived based on the ratio of telomere length between the sample and single copy gene standard (T/S ratio). The mean ratio of placental telomere in term live births was 5.181 ± 3.841.A twofold decrease in telomere length was seen in SBs (over all 2.455 ± 1.239; p0.001). For early SBs (above 34 weeks), the T/S was 2.8884 ± 1.224 and for late SBs, the T/S was 2.207 ± 1.201, both lower than term live births (both p0.01). T/S remained lower both in small for gestational age-SB (2.639 ± 1.619) and appropriate for gestational age-SB (2.653 ± 1.335) with no difference between these subgroups (p = ns). T/S was lower in SB compared with spontaneous preterm births (PTBs) (6.382 ± 5.525; p0.01), whereas SBs telomere length were similar to those of preterm premature rupture of membranes (pPROM) (3.296 ± 3.599; p = ns).Substantial reduction in telomere length in SBs is indicative of placental senescence. These data provide mechanistic insights that premature aging may lead to placental dysfunction as an initiator of fetal demise in unexplained SBs.

Early cell death is a feature of neurodegenerative disorders. Telomere shortening is related to premature cellular senescence and could be a marker for cellular pathology in neurological diseases. Relative telomere length in dementia (N=70), Huntington's disease (N=35), ataxia telangiectasia (N=9), and age-group matched control samples (N=105) was measured as relative telomere copy/single copy gene ratios. Individuals with Huntington's disease had the lowest relative telomere copy/single copy gene ratio (0.21), followed by ataxia telangiectasia (0.31) and dementia (0.48). The younger control group had the highest relative telomere copy/single copy gene ratio (1.07). The reduced telomere length could be indicative of shared biological pathways across these disorders contributing to cellular senescence.

Background: \ud Both active smoking and second-hand smoke (SHS) are important risk factors for many age-related diseases. Active smoking is associated with shortened telomere length. However, whether SHS accelerates telomere attrition with age is uncertain. The aim of this study was to examine the association between SHS exposure and shortening by age of leukocyte telomere length among adult non-smokers.\ud Methods: \ud We undertook a cross-sectional study of the association between self-reported levels of SHS exposure and telomere length shortening per annum on a subgroup of participants from the Scottish Family Health Study. Inclusion was restricted to non-smokers aged ≥ 18 years, who had provided self-reported overall usual SHS exposure (total hours per week) and blood samples for telomere analysis. Linear regression models were used to compare the ratio of telomere repeat copy number to single copy gene number (T/S)by age according to SHS exposure.\ud Results: \ud Of the 1303 eligible participants, 779 (59.8%) reported no SHS exposure, 495 (38.0%) low exposure (1–19 h per week) and 29 (2.2%) high exposure (≥20 h per week). In the univariate linear regression analyses, relative T/S ratio declined with increasing age in all exposure groups. Telomere length decreased more rapidly with increasing age among those with high exposure to SHS [adjusted coefficient −0.019, 95% confidence interval (CI) −0.031- −0.007) when compared with both those with no exposure to SHS (adjusted coefficient −0.006, 95% CI −0.008- −0.004) (high vs no SHS: P = 0.010) and those with low exposure to SHS (adjusted coefficient −0.005, 95% CI −0.007- −0.003) (high vs low SHS: P = 0.005).\ud Conclusions: \ud Our findings suggest that high SHS exposure may accelerate normal biological ageing, and support efforts to protect the public from SHS exposure. Further studies on relevant mechanisms should be conducted.

Background: The age-dependent increase of COPD prevalence suggests an intimate relationship between the process of aging and the development of COPD. Although it remains incompletely understood how the aging process and COPD are interrelated, some previous studies reported the role of telomere length shortening. We aimed to investigate the association between telomere length and COPD as well as its correlation with CT-measured emphysema severity. Method: We measured telomere length of peripheral blood mononuclear cells using a real-time polymerase chain reaction-based method in 150 patients with COPD and 102 control subjects with normal lung function. The telomere lengths were standardized to the reference single copy gene (T/S). CT-determined emphysema index (percentage area of lung with attenuation values below -950HU) and wall area percent (WA%) were also assessed. Results: Mean telomere length (T/S ratio) was significantly lower in patients with COPD than in control (0.46 ± 0.44 vs 0.94 ± 0.49; p Conclusion: Telomere shortening is associated with COPD and emphysema severity. These results support that accelerated aging is of particular relevance to COPD.

Emerging evidence suggests that epigenetics regulates telomere dynamics in adults. However, the relationship between these pathways in children and youth remains unknown. Thus, we examined this association in 542 healthy adolescents aged 14 to 18 years old (44.8% African Americans; 55.2% females). Global DNA methylation level (%5-mC) was quantified using ELISA method. Leukocyte telomere length (LTL) was defined as relative telomere to single copy gene (T/S) ratio. Multiple linear regression models, adjusted for age, gender, ethnicity, Tanner stage, BMI, PA, and batch effect, revealed that %5 mC was associated with LTL (adjusted β = 0.17, p 0.01). %5 mC accounted for 5.0% of the variation for LTL. A significant gender interaction was identified (p 0.01). There was an association between %5 mC and LTL in females (all ps ps

In crime investigation, some 40% of the DNA profiles recovered from the crime scene do not match to DNA profiles in the criminal DNA database. Hence, the ability to predict age from DNA samples provides additional information that would facilitate crime investigations. The present study aimed to develop a correlation model between signal joint T-cell receptor excision circle (sjTREC) quantity and age. A dual-TaqMan ® probe qPCR assay was used to quantify sjTREC numbers, normalised against the single copy gene albumin. This novel dual-probe assay showed an increased sensitivity as sjTREC could be amplified in donors up to 67 years old using only 12ng of DNA with less than 40 PCR cycles. In comparison, literature had cited at least 50ng of DNA with more than 40 PCR cycles. The age prediction accuracy of ±8.6 years for the Singapore Chinese population was comparable to previous studies. The results also suggested that the trend of sjTREC numbers with age could be different between the Singapore Chinese and Malay populations. However, a larger Malay sample size would be required to ascertain this observation.

The tandemly repeated hexamers (TTAGGG)n present at the telomeric ends protect the human genome from a variety of environmental exposures including ionizing radiation. In order to find out the effect of chronic low dose radiation exposure, we have determined telomere length among newborns from high level natural radiation areas (HLNRA) of the Kerala coast in South west India.Umbilical cord blood samples were collected from 128 newborns from HLNRA and 43 newborns from normal level natural radiation areas (NLNRA) and genomic DNA was isolated using a salt precipitation method. The mean telomere length was determined using SYBR green-based real time quantitative Polymerase chain reaction (qPCR), where telomere gene specific (T) and single copy gene specific (S) primers were used. The average of telomere versus single copy gene (T/S) ratio was calculated which was proportional to telomere length of each individual.The mean relative telomere length was found to be 1.03 ± 0.01 (95% CI, 0.99-1.05) and 1.10 ± 0.03 (95% CI, 1.04-1.17) in HLNRA and NLNRA newborns, respectively (P 0.05). Based on the level of background radiation dose, samples were divided into four groups, i.e., NLNRA: 1.50 mGy/year and three HNLRA groups: 1.51-3.00 mGy/year, 3.01-5.00 mGy/year, and 5.00 mGy/year. The mean relative telomere length in these groups were found to be 1.10 ± 0.03 (95% CI, 1.03-1.17), 0.98 ± 0.01 (95% CI, 0.95-1.01), 1.05 ± 0.02 (95% CI, 1.01-1.10) and 1.07 ± 0.03 (95% CI, 1.04-1.10), respectively. No significant difference was observed between the mean telomere length of male and female newborns.The elevated level of natural chronic background radiation prevailing in Kerala coast did not show any significant effect on telomere length of newborns. To our knowledge, this is the first report on newborn telomere length from a high level natural radiation area.

Association Between Telomere Shortening and Ageing During Occupational ExposureTelomere length is considered as a biomarker of ageing, resulting in shortening during the process. The present investigation was an attempt to determine the relative telomere length in mechanical workshop workers. Telomere length shortening in cells during occupational exposure causes accelerated ageing. Genomic DNA was isolated from buccal epithelial cells collected from 240 individuals, comprising two groups of 120 exposed workers and 120 unexposed controls. Telomere length was measured by using real time PCR. Both telomere (T) and single copy gene (S) specific primers were used to compute the relative T/S ratio and expressed as the relative telomere length. Telomere length differed significantly between the workers and controls (pp

Amh is a single copy gene which is expressed in different ways during mammalian development. Several potential promoter elements have been identified using physiological experimentation and on the basis of interspecific sequence comparison. The role of putative promoter elements in controlling gene expression has been investigated by many workers over the last two decades and here by individually mutating each element. Expression was measured in vitro in cells of Sertoli descent by flowcytometry using EGFP as a reporter gene. Three lines of murine cells were used; pre- and post-pubertal Sertoli and granulosa cells. Differences between the three lines of cells, support the view that differentiation in this in vitro model system is likely to be at the level of available transcription factors at given points in development.

Discrimination of homologous and heterozygous plants at the male-determination locus in asparagus (Asparagus officinalis L.) was examined by quantitative analyses of a diagnostic marker for male asparagus. The quantitative difference of a sequence, AspTaq1, derived from the Asp1-T7 diagnostic marker (Jamsari et al., 2004), was measured using the real-time monitoring PCR and normalized based on the quantitative difference of a sequence, AODEF-Taq1, derived from a single copy gene, AODEF. The normalized difference was higher in established supermales than in regular males grown from commercially available seeds. The averaged values in respective groups are in the approximate ratio of 2 : 1. Homozygous male (MM), heterozygous male (Mm), and female (mm) genotypes were obtained by selfing of an andromonoecious line. The progeny were divided into three groups by the normalized difference; plants showing high values and intermediate values were thought to be homozygous and heterozygous males, respectively. Significant amplification of AspTaq1 was not detected in several plants; therefore, they were judged to be females. Then, to identify supermales, pollen from male plants was crossed separately with female plants, and the sex of the next generation was determined. Pollen from two male plants showing high values of the normalized difference generated only males in the next generation, while pollen from 12 male plants showing intermediate values generated both female and male plants. Pollen from one male plants that showed intermediate values generated only male plants. The normalized difference in homozygous males was comparable to the value obtained using a recombinant sequence that contains an equal number of AspTaq1 and AODEF-Taq1. These results indicate that homozygous and heterozygous males can be identified by quantitative analysis of a nucleotide sequence linked to the sex-determination locus and that the use of an authentic sequence for calibration facilitates the discrimination process of those genotypes in asparagus plants.

Objective Reduced telomere length may be a marker of biological aging. We hypothesized that telomere length might thus relate to increased risk for dementia and mortality. Methods This nested case–control study used stored leukocyte DNA from 257 individuals (mean age, 81.4 ± 7.9 years; 64.6% female; 44.7% Hispanic, 33.5% non-Hispanic black, and 21.8% non-Hispanic white). Our assay used real-time polymerase chain reaction, with two separate reactions amplifying telomere sequence and reference single copy gene (ribosomal-protein-P0), providing a calculated telomere-to-single copy gene (T/S) ratio. Results Mean telomere length was shorter among subjects dying during follow-up than in those surviving (0.453 ± 0.211 vs 0.525 ± 0.226 [± standard deviation]; p < 0.009). It was also shorter in those with Alzheimer's disease compared with control subjects (0.458 ± 0.207 vs 0.516 ± 0.229; p < 0.03). For participants with Alzheimer's disease, compared with those with the longest telomeres, the mortality odds ratio (OR) was 4.8 (95% confidence interval [CI], 1.7–13.8) in those with intermediate-length telomeres and 7.3 (95% CI, 2.4–22.0) in those with the shortest telomeres. The presence of an e4 allele also increased the mortality OR, with an OR of 5.8 (95% CI, 1.3–26.4) for intermediate-length telomeres and an OR of 9.0 (95% CI, 1.9–41) for the shortest telomeres. Interpretation Our findings suggest that leukocyte telomere length is related to both dementia and mortality and may be a marker of biological aging. Ann Neurol 2006

A method is presented for the quantification of haddock in complex food matrices based on real-time PCR. The proportion of haddock muscle tissue to the numbers of a single copy gene has been shown to remain relatively constant, throughout the year and across a number of fishing grounds, using conventional muscle nitrogen content as a comparator. This indicates that quantification of fish, based upon nitrogen content or the numbers of a single copy gene, would return data with a similar degree of accuracy. Thus, a haddock-specific, real-time PCR primer and probe set was designed and optimised, and when used in conjunction with a haddock calibration curve was shown to be able to quantify model samples to within 7% of the true percentage. This is the first report of a real-time assay for the quantification of white fish, which would allow enforcement of the legislation in a complex food matrix containing haddock with other fish species.

ATBP [(A+T)-stretch binding protein] activates the Sarcophaga defense protein genes in an (A+T)-stretch dependent manner as shown previously using a luciferase reporter assay [Aozasa et al. (2001) Eur J Biochem, 268, 2506-2511]. We identified the Drosophila homologue of ATBP. Drosophila ATBP has 388 amino acid residues and the amino acid sequence has high similarity with that of the Sarcophaga ATBP. In particular, residues 1-56 and 301-374 are highly conserved between Sarcophaga and Drosophila. Drosophila ATBP activated the Sarcophaga lectin gene promoter in SL-2 cells. The Drosophila ATBP gene is a single copy gene and is located in the 20E region of chromosome X.

We investigated 31 samples of charred archaeological cereal grains from the Northern Alpine region, dating from Neolithic to medieval times. One hundred and twenty five DNA extracts from 23 extraction series were screened for the presence of authentic DNA by PCR amplification of a 240 bp fragment from the high molecular weight glutenin subunit gene promoter (HGP) region. Criteria of authenticity adjusted to the features of charred cereal remains were applied. No PCR products were amplified in most extracts. Extraction series with positive results were further analysed with additional primer sets directed at the single-copy HGP region and with primers for the ribosomal (ITS2) or the chloroplast ( rbcL ) DNA. Most positive results of the HGP regions were subsequently considered not authentic, mainly because of molecular inconsistency. In four extracts the successful amplification of the rbcL region, the ITS2 region, or the HGP and the ITS2 region suggested the presence of authentic DNA but further and indep...

Background Single copy genes are common across angiosperm genomes. With the sufficiently high quality sequenced genomes, the identification of large-scale single copy genes among multiple species is possible. Although some characteristics have been reported, our study provides novel insights into single copy genes. Results We identified single copy genes across 29 angiosperm genomes. A significant negative correlation was found between the number of duplicate blocks and the number of single copy genes. We found that a considerable number of single copy genes are located in organelles, showing a preference for binding and catalytic activity. The analysis of effective number of codons (Nc) illustrates that single copy genes have a stronger codon bias than non-single copy genes in eudicots. The relative high expression level of single copy genes was partially confirmed by the RNA-seq data, rather than the Codon Adaptation Index (CAI). Unlike in most other species, a strongly negatively correlation occurs between Nc and GC3 among single copy genes in grass genomes. When compared to all non-single copy genes, single copy genes indicate more conservation (as indicated by Ka and Ks values). But our alternative splicing (AS) results reveal that selective constraints are weaker in single copy genes than in low copy family genes (1–10 in-paralogs) and stronger than high copy family genes (>10 in-paralogs). Using concatenated shared single copy genes, we obtained a well-resolved phylogenetic tree. With the addition of intron sequences, the branch support is improved, but striking incongruences are also evident. Therefore, it is noteworthy that inclusion of intron sequences seems more appropriate for the phylogenetic reconstruction at lower taxonomic levels. Conclusions Our analysis provides insight into the evolutionary characteristics of single copy genes across 29 angiosperm genomes. The results suggest that there are key differences in evolutionary constraints between single copy genes and non-single copy genes. And to some extent, these evolutionary constraints show some species-specific differences, especially between eudicots and monocots. Our preliminary evidence also suggests that the concatenated shared single copy genes are well suited for use in resolving phylogenetic relationships. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-504) contains supplementary material, which is available to authorized users.

The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland.

espanolIntroduccion: Los telomeros son estructuras de los cromosomas eucariotas, que juegan un importante papel en el mantenimiento de la estabilidad e integridad celular, estableciendose como uno de los posibles biomarcadores del envejecimiento celular en el hombre y otras especies. Objetivos: Evaluar la posibilidad del uso de la tecnica de la reaccion en cadena de la polimerasa cuantitativa (qPCR) para calcular la longitud relativa de los telomeros en caballos y asociarla con factores como la edad, la raza y el y el sexo. Material y metodos: Se amplifico una secuencia repetitiva correspondiente a los telomeros, junto con otra de un gen de copia unica (Interleukina-2), en muestras de sangre de 175 caballos de tres razas, de distintos grupos de edad y de ambos sexos. Se calculo la longitud relativa de los telomeros de cada individuo a traves dos variables de respuesta: la diferencia entre el numero de ciclos de cuantificacion entre telomeros e Interleukina-2 (Dif Cq) y la ratio entre ambos valores (T/S ratio). Resultados: La variable Dif Cq presenta una asociacion mas fuerte con la edad que la variable T/S ratio. Se observo una correlacion negativa entre la edad y la longitud de los telomeros (R= -0,31, p EnglishIntroduction: Telomeres are specialized structures at the ends of eukaryotic chromosomes which play an important role in the maintenance of cell integrity and stability. The telomere length is considered one of the potential biomarkers of cellular aging. Objectives: To evaluate the possible use of quantitative polymerase chain reaction qPCR in the estimation of relative length of equine chromosome telomeres. Material and methods: A repetitive sequence corresponding to telomeres and a single copy gene (Interleukin-2) were amplified from blood samples of 175 horses of three breeds, different ages and both sexes. The relative length of telomeres of each individual was calculated by means of the response variables: the difference of quantification cycles number between telomeres and Interleukin-2 (Dif Cq) and the ratio between both values (T/S ratio). Results: The variable Dif Cq has a stronger association with the age than the variable T/S ratio. There was a negative correlation between the age and telomere length (R= -0,31, p

Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.