bluuebunny/crossref_metadata_2025 huggingface dataset:
Multimodal regularized linear models with flux balance analysis for mechanistic integration of omics data
Authors: Magazzù Giuseppe , Zampieri Guido , Angione Claudio
Published: April 2021

Motivation

High-throughput biological data, thanks to technological advances, have become cheaper to collect, leading to the availability of vast amounts of omic data of different types. In parallel, the in silico reconstruction and modeling of metabolic systems is now acknowledged as a key tool to complement experimental data on a large scale. The integration of these model- and data-driven information is therefore emerging as a new challenge in systems biology, with no clear guidance on how to better take advantage of the inherent multisource and multiomic nature of these data types while preserving mechanistic interpretation.

Results

Here, we investigate different regularization techniques for high-dimensional data derived from the integration of gene expression profiles with metabolic flux data, extracted from strain-specific metabolic models, to improve cellular growth rate predictions. To this end, we propose ad-hoc extensions of previous regularization frameworks including group, view-specific and principal component regularization and experimentally compare them using data from 1143 Saccharomyces cerevisiae strains. We observe a divergence between methods in terms of regression accuracy and integration effectiveness based on the type of regularization employed. In multiomic regression tasks, when learning from experimental and model-generated omic data, our results demonstrate the competitiveness and ease of interpretation of multimodal regularized linear models compared to data-hungry methods based on neural networks.

Availability and implementation

All data, models and code produced in this work are available on GitHub at https://github.com/Angione-Lab/HybridGroupIPFLasso_pc2Lasso.

Supplementary information

Supplementary data are available at Bioinformatics online.
Assessment of significance of conditionally independent GWAS signals
Authors: Ghasemi Sahar , Teumer Alexander , Wuttke Matthias , Becker Tim
Published: April 2021

Motivation

Multiple independently associated SNPs within a linkage disequilibrium region are a common phenomenon. Conditional analysis has been successful in identifying secondary signals. While conditional association tests are limited to specific genomic regions, they are benchmarked with genome-wide scale criterion, a conservative strategy. Within the weighted hypothesis testing framework, we developed a ‘quasi-adaptive’ method that uses the pairwise correlation (r2) and physical distance (d) from the index association to construct priority functions G =G(r2, d), which assign an SNP-specific α-threshold to each SNP. Family-wise error rate (FWER) and power of the approach were evaluated via simulations based on real GWAS data. We compared a series of different G-functions.

Results

Simulations under the null hypothesis on 1,100 primary SNPs confirmed appropriate empirical FWER for all G-functions. A G-function with optimal r2 = 0.3 between index and secondary SNP which down-weighted SNPs at higher distance step-wise-strong and gave more emphasis on d than on r2 had overall best power. It also gave the best results in application to the real datasets. As a proof of concept, ‘quasi-adaptive’ method was applied to GWAS on free thyroxine (FT4), inflammatory bowel disease (IBD) and human height. Application of the algorithm revealed 5 secondary signals in our example GWAS on FT4, 5 secondary signals in case of the IBD and 19 secondary signals on human height, that would have gone undetected with the established genome-wide threshold (α=5×10−8).

Availability and implementation

https://github.com/sghasemi64/Secondary-Signal.

Supplementary information

Supplementary data are available at Bioinformatics online.
Study of real-valued distance prediction for protein structure prediction with deep learning
Authors: Li Jin , Xu Jinbo
Published: April 2021

Motivation

Inter-residue distance prediction by convolutional residual neural network (deep ResNet) has greatly advanced protein structure prediction. Currently, the most successful structure prediction methods predict distance by discretizing it into dozens of bins. Here, we study how well real-valued distance can be predicted and how useful it is for 3D structure modeling by comparing it with discrete-valued prediction based upon the same deep ResNet.

Results

Different from the recent methods that predict only a single real value for the distance of an atom pair, we predict both the mean and standard deviation of a distance and then fold a protein by the predicted mean and deviation. Our findings include: (i) tested on the CASP13 FM (free-modeling) targets, our real-valued distance prediction obtains 81% precision on top L/5 long-range contact prediction, much better than the best CASP13 results (70%); (ii) our real-valued prediction can predict correct folds for the same number of CASP13 FM targets as the best CASP13 group, despite generating only 20 decoys for each target; (iii) our method greatly outperforms a very new real-valued prediction method DeepDist in both contact prediction and 3D structure modeling and (iv) when the same deep ResNet is used, our real-valued distance prediction has 1–6% higher contact and distance accuracy than our own discrete-valued prediction, but less accurate 3D structure models.

Availability and implementation

https://github.com/j3xugit/RaptorX-3DModeling.

Supplementary information

Supplementary data are available at Bioinformatics online.
Model-Based Geostatistical Methods Enable Efficient Design and Analysis of Prevalence Surveys for Soil-Transmitted Helminth Infection and Other Neglected Tropical Diseases
Authors: Johnson Olatunji , Fronterre Claudio , Amoah Benjamin , Montresor Antonio , Giorgi Emanuele , Midzi Nicholas , Mutsaka-Makuvaza Masceline Jenipher , Kargbo-Labor Ibrahim , Hodges Mary H , Zhang Yaobi , Okoyo Collins , Mwandawiro Charles , Minnery Mark , Diggle Peter J
Published: April 2021

Maps of the geographical variation in prevalence play an important role in large-scale programs for the control of neglected tropical diseases. Precontrol mapping is needed to establish the appropriate control intervention in each area of the country in question. Mapping is also needed postintervention to measure the success of control efforts. In the absence of comprehensive disease registries, mapping efforts can be informed by 2 kinds of data: empirical estimates of local prevalence obtained by testing individuals from a sample of communities within the geographical region of interest, and digital images of environmental factors that are predictive of local prevalence. In this article, we focus on the design and analysis of impact surveys, that is, prevalence surveys that are conducted postintervention with the aim of informing decisions on what further intervention, if any, is needed to achieve elimination of the disease as a public health problem. We show that geospatial statistical methods enable prevalence surveys to be designed and analyzed as efficiently as possible so as to make best use of hard-won field data. We use 3 case studies based on data from soil-transmitted helminth impact surveys in Kenya, Sierra Leone, and Zimbabwe to compare the predictive performance of model-based geostatistics with methods described in current World Health Organization (WHO) guidelines. In all 3 cases, we find that model-based geostatistics substantially outperforms the current WHO guidelines, delivering improved precision for reduced field-sampling effort. We argue from experience that similar improvements will hold for prevalence mapping of other neglected tropical diseases.
Severe Acute Respiratory Syndrome Coronavirus 2 Infection Among Healthcare Workers in South Africa: A Longitudinal Cohort Study
Authors: Nunes Marta C , Baillie Vicky L , Kwatra Gaurav , Bhikha Sutika , Verwey Charl , Menezes Colin , Cutland Clare L , Moore David P , Dangor Ziyaad , Adam Yasmin , Mathivha Rudo , Velaphi Sithembiso C , Tsitsi Merika , Aguas Ricardo , Madhi Shabir A ,   Bara HCW Study Group, Nakwa Firdose , Van Blydenstein Sarah , Venter Michelle , Reddy Denasha , du Plessis Jeanine , Laubscher Matt , van der Merwe Lara , Mbele Nkululeko , Mukendi Beya , McKenzie Shakeel , Mtshali Sihle , Mukendi Christian Kabasele , Nzimande Ayanda , Mandindi Wendy Zimkhitha , Nana Amit Jawaharlal , Rafuma Martin Mosotho , Mathibe Masego Nicole , Moultrie Andrew
Published: April 2021

From April to September 2020, we investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in a cohort of 396 healthcare workers (HCWs) from 5 departments at Chris Hani Baragwanath Hospital, South Africa. Overall, 34.6% of HCWs had polymerase chain reaction–confirmed SARS-CoV-2 infection (132.1 [95% confidence interval, 111.8–156.2] infections per 1000 person-months); an additional 27 infections were identified by serology. HCWs in the internal medicine department had the highest rate of infection (61.7%). Among polymerase chain reaction–confirmed cases, 10.4% remained asymptomatic, 30.4% were presymptomatic, and 59.3% were symptomatic.
Functional megalin is expressed in renal cysts in a mouse model of adult polycystic kidney disease
Authors: Nielsen Marlene L , Mundt Mia C , Lildballe Dorte L , Rasmussen Maria , Sunde Lone , Torres Vicente E , Harris Peter C , Birn Henrik
Published: April 2021

Background

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive growth of cysts and a decline of renal function. The clinical feasibility of the number of potential disease-modifying drugs is limited by systemic adverse effects. We hypothesize that megalin, a multiligand endocytic receptor expressed in the proximal tubule, may be used to facilitate drug uptake into cysts, thereby allowing for greater efficacy and fewer side effects.

Methods

The cyst expression of various tubular markers, including megalin and aquaporin 2 (AQP2), was analysed by immunohistochemistry (IHC) of kidney sections from the ADPKD mouse model (PKD1RC/RC) at different post-natal ages. The endocytic function of megalin in cysts was examined by IHC of kidney tissue from mice injected with the megalin ligand aprotinin.

Results

Cyst lining epithelial cells expressing megalin were observed at all ages; however, the proportion decreased with age. Concomitantly, an increasing proportion of cysts revealed expression of AQP2, partial expression of megalin and/or AQP2 or no expression of the examined markers. Endocytic uptake of aprotinin was evident in megalin-positive cysts, but only in those that remained connected to the renal tubular system.

Conclusions

Megalin-expressing cysts were observed at all ages, but the proportion decreased with age, possibly due to a switch in tubular origin, a merging of cysts of different tubular origin and/or a change in the expression pattern of cyst lining cells. Megalin expressed in cysts was functional, suggesting that megalin-mediated endocytosis is a potential mechanism for drug targeting in ADPKD if initiated early in the disease.
Performance of a Paired-End Sequencing-Based Noninvasive Prenatal Screening Test in the Detection of Genome-Wide Fetal Chromosomal Anomalies
Authors: Pertile Mark D , Flowers Nicola , Vavrek Darcy , Andrews Daniel , Kalista Tasha , Craig Andrew , Deciu Cosmin , Duenwald Sven , Meier Kristen , Bhatt Sucheta
Published: April 2021

Background

Noninvasive prenatal tests (NIPTs) detect fetal chromosomal anomalies with high clinical sensitivity and specificity. We examined the performance of a paired-end sequencing-based NIPT in the detection of genome-wide fetal chromosomal anomalies including common trisomies, sex chromosomal aneuploidies (SCA), rare autosomal aneuploidies (RAAs), and partial deletions/duplications ≥7 Mb.

Methods

Frozen plasma samples from pregnant women were tested using the VeriSeq NIPT Solution v2 assay. All samples were previously tested with a laboratory-developed NIPT and had known clinical outcomes. Individuals performing the sequencing were blinded to clinical outcome data. Clinical sensitivity and specificity were determined for basic (chromosomes 21, 18, 13, X, and Y) and genome-wide screening modes.

Results

Of 2335 samples that underwent genome-wide analysis, 28 did not meet QC requirements, resulting in a first-pass assay failure rate of 1.2%. Basic screening analysis, excluding known mosaics, correctly classified 130/130 trisomy 21 samples (sensitivity >99.9%, 95% confidence interval [CI] 97.1%–100%), 41/41 trisomy 18 samples (sensitivity >99.9%, 95% CI 91.4%–100%), and 26/26 trisomy 13 samples (sensitivity >99.9%, 95% CI 87.1%–100%) with 6 false-positive results; specificities ≥99.90% were reported for all 3 trisomies. Concordance for SCAs ranged from 90.5%–100%. Genome-wide screening analysis including known mosaics correctly classified 27/28 RAAs and 20/27 partial deletions/duplications with a specificity of 99.80% for both anomalies, and an overall genome-wide specificity for all anomalies of 99.34%.

Conclusions

The VeriSeq NIPT Solution v2 assay enables accurate identification of fetal aneuploidy, allowing detection of genome-wide fetal chromosomal anomalies with high clinical sensitivities and specificities and a low assay failure rate.

Clinical Trial Notification [CTN] identification number [ID]: CT-2018-CTN-01585-1 v1, Protocol: NIPT T05 002.
Robotic thymectomy for thymoma in patients with myasthenia gravis: neurological and oncological outcomes
Authors: Romano Gaetano , Zirafa Carmelina Cristina , Ceccarelli Ilaria , Guida Melania , Davini Federico , Maestri Michelangelo , Morganti Riccardo , Ricciardi Roberta , Hung Key Teresa , Melfi Franca
Published: April 2021

OBJECTIVES

The goal of this study was to analyse the outcomes in 53 patients with thymoma, 34 of whom had myasthenia gravis (MG), who were treated with robotic surgery. The oncological outcomes of the whole series of patients were analysed. Furthermore, because consistent data are not yet available in the literature, the main focus was the analysis of the neurological results of the patients affected by MG and thymoma.

METHODS

The clinical outcomes of 53 patients with a diagnosis of thymoma who underwent robotic thymectomy between January 2014 and December 2019 in our institution were collected and evaluated; 34 of these patients had a concomitant diagnosis of MG. The neurological status of the patients was determined from a clinical evaluation according to the Osserman classification and on pre- and post-surgery Myasthenia Gravis Composite scores, whereas neurological clinical outcomes were assessed using the Myasthenia Gravis Foundation of America Post-Intervention Score. Reduction of steroid therapy was also considered. The recurrence rate, adjuvant radiotherapy and overall survival of the patients with a thymoma were evaluated.

RESULTS

Neurological outcomes: improvement of the clinical conditions was obtained in 26 patients (76.5%) following the operation: complete stable remission was observed in 5 patients (14.7%), pharmacological remission in 10 (29.4%) and minimal manifestation in 11 (32.3%). Four patients (11.8%) exhibited no substantial change from the pretreatment clinical manifestations or reduction in MG medication and 4 (11.8%) patients experienced worsening of clinical conditions. In 21 patients (61.7%) a reduction of the dosage of steroid therapy was obtained. Oncological outcomes: at an average follow-up of 36 months, the overall survival was 96%, 4 patients (7.5%) had pleural relapses and 12 patients (22.6%) underwent postoperative radiotherapy, according to their stage. In accordance with Masaoka staging, 34% were in stage I, 56.6% in stage II and 9.4% in stage III

CONCLUSIONS

Our results suggest that robotic surgical treatment of patients with thymoma and concomitant MG is effective in improving the neurological outcomes. Moreover, the oncological results obtained in this series confirm the efficacy of robotic surgery for the treatment of thymic malignancies, with results in line with those of open surgery. However, due to the indolent growth of thymomas, further observations with longer follow-up are necessary.
Isolation and identification of a human intestinal bacterium capable of daidzein conversion
Authors: Guo Yingyu , Zhao Lichao , Fang Xiang , Zhong Qingping , Liang Huijun , Liang Wenou , Wang Li
Published: April 2021

Equol, which produced from daidzein (one of the principal isoflavones), is recognized to be the most resultful in stimulating an estrogenic and antioxidant response. The daidzein transformation was studied during fermentation of five growth media inoculated with feces from a healthy human, and a daidzein conversion strain was isolated. To enrich the bacterial population involved in daidzein metabolism in a complex mixture, fecal samples were treated with antibiotics. The improved propidium monoazide combined with the quantitative polymerase chain reaction (PMAxx-qPCR) assay showed that the ampicillin treatment of samples did result in a reduction of the total visible bacteria counts by 52.2% compared to the treatment without antibiotics. On this basis, the newly isolated rod-shaped, Gram-positive anaerobic bacterium, named strain Y11 (MN560033), was able to metabolize daidzein to equol under anaerobic conditions, with a conversion ratio (equol ratio: the amount of equol produced/amount of supplemented daizein) of 0.56 over 120 h. The 16S rRNA partial sequence of the strain Y11 exhibited 99.8% identity to that of Slackia equolifaciens strain DZE (NR116295). This study will provide new insights into the biotransformation of equol from daidzein by intestinal microbiota from the strain-level and explore the possibility of probiotic interventions.
Preliminary Evidence that Intraspecific Competition Increases Size of Restoration-Planted Pitch and Shortleaf Pines in a Mixed-Hardwood Clearcut in the Southern Appalachians
Authors: McNab W Henry
Published: April 2021

Oak-pine (Quercus L. - Pinus L.) forest communities on low ridges in the southern Appalachian Mountains are losing diversity as mature pitch (P. rigida Mill.) and shortleaf (P. echinata Mill.) pines die and do not regenerate under a hardwood canopy. Restoration of biodiversity by planting pine seedlings is well known, but little is known regarding whether the configuration of planted seedlings affects growth and subsequent size (diameter at breast height, dbh) as trees age. The purpose of this study was to test the hypothesis that pines growing in groups of two or more trees respond with increased growth (expressed by dbh) to intraspecific competition with other pines compared to single trees subjected only to interspecific competition with surrounding hardwoods. For 13-year-old pitch and shortleaf pines, trees were larger in dbh when occurring in groups than trees occurring singly. Regression indicated that intraspecific competition accounted for 16% of the dbh variation of pitch pine and 29% for shortleaf pine. This study originated from chance observations in a small study of pine restoration. If a designed study confirms these results, resource managers could restore biodiversity with reduced site disturbance and establishment costs by planting pine seedlings in small groups rather than rows.
Gene Coexpression Network Reveals Highly Conserved, Well-Regulated Anti-Ageing Mechanisms in Old Ant Queens
Authors: Harrison Mark C , Niño Luisa M Jaimes , Rodrigues Marisa Almeida , Ryll Judith , Flatt Thomas , Oettler Jan , Bornberg-Bauer Erich
Published: April 2021

Evolutionary theories of ageing predict a reduction in selection efficiency with age, a so-called “selection shadow,” due to extrinsic mortality decreasing effective population size with age. Classic symptoms of ageing include a deterioration in transcriptional regulation and protein homeostasis. Understanding how ant queens defy the trade-off between fecundity and lifespan remains a major challenge for the evolutionary theory of ageing. It has often been discussed that the low extrinsic mortality of ant queens, that are generally well protected within the nest by workers and soldiers, should reduce the selection shadow acting on old queens. We tested this by comparing strength of selection acting on genes upregulated in young and old queens of the ant, Cardiocondyla obscurior. In support of a reduced selection shadow, we find old-biased genes to be under strong purifying selection. We also analyzed a gene coexpression network (GCN) with the aim to detect signs of ageing in the form of deteriorating regulation and proteostasis. We find no evidence for ageing. In fact, we detect higher connectivity in old queens indicating increased transcriptional regulation with age. Within the GCN, we discover five highly correlated modules that are upregulated with age. These old-biased modules regulate several antiageing mechanisms such as maintenance of proteostasis, transcriptional regulation, and stress response. We observe stronger purifying selection on central hub genes of these old-biased modules compared with young-biased modules. These results indicate a lack of transcriptional ageing in old C. obscurior queens, possibly facilitated by strong selection at old age and well-regulated antiageing mechanisms.
Fine-Scale Spatial Genetic Structure with Nonuniform Distribution of Individuals
Authors: Doligez Agnès , Baril Claire , Joly Hélène I
Published: April 2021

This paper presents the first theoretical study of spatial genetic structure within nonuniformly distributed continuous plant populations. A novel individual-based model of isolation by distance was constructed to simulate genetic evolution within such populations. We found larger values of spatial genetic autocorrelations in highly clumped populations than in uniformly distributed populations. Most of this difference was caused by differences in mean dispersal distances, but aggregation probably also produced a slight increase in spatial genetic structure. Using an appropriate level of approximation of the continuous distribution of individuals in space, we assessed the potential effects of density, seed and pollen dispersal, generation overlapping, and overdominance selection at an independent locus, on fine-scale genetic structure, by varying them separately in a few particular cases with extreme clumping. When selfing was allowed, all these input variables influenced both aggregation and spatial genetic structure. Most variations in spatial genetic structure were closely linked to variations in clumping and/or local density. When selfing was not allowed, spatial genetic structure was lower in most cases.
Isolation of the GA-Response Mutant sly1 as a Suppressor of ABI1-1 in Arabidopsis thaliana
Authors: Steber Camille M , Cooney Sarah E , McCourt Peter
Published: April 2021

Seed dormancy and germination in higher plants are partially controlled by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). ABA establishes dormancy during embryo maturation, whereas GA breaks dormancy and induces germination. Previous attempts to identify GA response genes were confounded because GA mutants are not expected to germinate and, unlike GA auxotrophs, should fail to be rescued by exogenous GA. Here, we describe a screen for suppressors of the ABA-insensitive mutant ABI1-1 that enriches for GA auxotrophs and GA-insensitive mutants. The vast majority (76%) of the suppressors of ABI1-1 strongly resemble GA auxotrophs in that they are severely dwarfed and have dark green foliage and flowers with underdeveloped petals and stamen. Three isolates were alleles of the GA auxotroph ga1. The remaining severe dwarves were not rescued by GA and belong to a single complementation group that we designate sly1 (Sleepy 1). The alleles of sly1 identified are the first recessive GA-insensitive mutations to reflect the full spectrum of GA-associated phenotypes, including the failure to germinate in the absence of the ABI1-1 lesion. Thus, we postulate that SLY1 is a key factor in GA reception.
Selection of T-DNA-Tagged Male and Female Gametophytic Mutants by Segregation Distortion in Arabidopsis
Authors: Howden Ross , Park Soon Ki , Moore James M , Orme James , Grossniklaus Ueli , Twell David
Published: April 2021

As a strategy for the identification of T-DNA-tagged gametophytic mutants, we have used T-DNA insertional mutagenesis based on screening for distorted segregation ratios by antibiotic selection. Screening of ~1000 transgenic Arabidopsis families led to the isolation of eight lines showing reproducible segregation ratios of ~1:1, suggesting that these lines are putative gametophytic mutants caused by T-DNA insertion at a single locus. Genetic analysis of T-DNA transmission through reciprocal backcrosses with wild type showed severe reductions in genetic transmission of the T-DNA through the male and/or female gametes. Direct evidence for mutant phenotypes in these lines was investigated by DAPI staining of mature pollen grains and by the analysis of seed set and embryo sac morphology in cleared ovules. One line, termed limpet pollen, showed a novel pollen phenotype in that the generative cell failed to migrate inward after pollen mitosis I, such that the generative or sperm cells remained against the pollen wall. Two other lines, andarta and tistrya, were defective in female transmission and showed an early arrest of embryo sac development with the viable megaspore not initiating the nuclear division cycles. These data demonstrate the efficacy of a segregation ratio distortion strategy for the identification of T-DNA-tagged gametophytic mutants in Arabidopsis.
Gene Silencing and Homology-Dependent Gene Silencing in Arabidopsis: Genetic Modifiers and DNA Methylation
Authors: Furner Ian J , Sheikh Mazhar A , Collett Clare E
Published: April 2021

Transgenes inserted into the plant genome can become inactive (gene silencing) or result in silencing of homologous cellular genes [homology-dependent gene silencing (HDG silencing)]. In an earlier study we reported HDG silencing of chalcone synthase (CHS) in Arabidopsis. This study concerns genetic revertants of one of the CHS HDG -silencing transgenic homozygotes. Two monogenic recessive trans-acting mutations (hog1 and ddm1) that impair gene silencing and HDG silencing were identified. These mutations reduce genomic DNA methylation and affect the quantity and size of CHS mRNA. These results imply that DNA methylation is necessary for both gene silencing and HDG silencing. Two further monogenic, trans-acting, recessive mutations (sil1 and sil2) reduce gene silencing but not HDG silencing. The existence of this mutant class shows that gene silencing involves genes that are not necessary for HDG silencing. A further mutant (Catt) was isolated and has an attenuated HDG-silencing T-DNA.
A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport
Authors: Jiang Yu , Scarpa Al , Zhang Li , Stone Shelly , Feliciano Ed , Ferro-Novick Susan
Published: April 2021

The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34°. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone α-factor at 37°. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.
Ion Tolerance of Saccharomyces cerevisiae Lacking the Ca2+/CaM-Dependent Phosphatase (Calcineurin) Is Improved by Mutations in URE2 or PMA1
Authors: Withee James L , Sen Romita , Cyert Martha S
Published: April 2021

Calcineurin is a conserved, Ca2+/CaM-stimulated protein phosphatase required for Ca2+-dependent signaling in many cell types. In yeast, calcineurin is essential for growth in high concentrations of Na+, Li+, Mn2+, and OH−, and for maintaining viability during prolonged treatment with mating pheromone. In contrast, the growth of calcineurin-mutant yeast is better than that of wild-type cells in the presence of high concentrations of Ca2+. We identified mutations that suppress multiple growth defects of calcineurin-deficient yeast (cnb1Δ or cna1Δ cna2Δ). Mutations in URE2 suppress the sensitivity of calcineurin mutants to Na+, Li+, and Mn2+, and increase their survival during treatment with mating pheromone. ure2 mutations require both the transcription factor Gln3p and the Na+ ATPase Pmr2p to confer Na+ and Li+ tolerance. Mutations in PMA1, which encodes the yeast plasma membrane H+-ATPase, also suppress many growth defects of calcineurin mutants. pma1 mutants display growth phenotypes that are opposite to those of calcineurin mutants; they are resistant to Na+, Li+, and Mn2+, and sensitive to Ca2+. We also show that calcineurin mutants are sensitive to aminoglycoside antibiotics such as hygromycin B while pma1 mutants are more resistant than wild type. Furthermore, pma1 and calcineurin mutations have antagonistic effects on intracellular [Na+] and [Ca2+]. Finally, we show that yeast expressing a constitutively active allele of calcineurin display pma1-like phenotypes, and that membranes from these yeast have decreased levels of Pma1p activity. These studies further characterize the roles that URE2 and PMA1 play in regulating intracellular ion homeostasis.
The Cloning by Complementation of the pawn-A Gene in Paramecium
Authors: Haynes W John , Vaillant Brian , Preston Robin R , Saimi Yoshiro , Kung Ching
Published: April 2021

The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.
Candidate Quantitative Trait Loci and Naturally Occurring Phenotypic Variation for Bristle Number in Drosophila melanogaster: The Delta-Hairless Gene Region
Authors: Lyman Richard F , Mackay Trudy F C
Published: April 2021

Delta (Dl) and Hairless (H) are two chromosome 3 candidate neurogenic loci that might contribute to naturally occurring quantitative variation for sensory bristle number. To evaluate this hypothesis, we assessed quantitative genetic variation in abdominal and sternopleural bristle numbers among homozygous isogenic third chromosomes sampled from nature and substituted into the Samarkand (Sam) inbred chromosome 1 and 2 background; among homozygous lines in which the wild-derived Dl-H gene region was introgressed into the Sam chromosome 3 background; and among Dl-H region introgression lines as heterozygotes against the Sam wild-type strain and derivatives of Sam into which mutant Dl and H alleles had been introgressed. Variation among the Dl-H region introgression lines accounted for 36% (8.3%) of the total chromosome 3 among line variance in abdominal (sternopleural) bristle number and for 53% of the chromosome 3 sex × line variance in abdominal bristle number. Naturally occurring alleles in the Dl-H region failed to complement a Dl mutant allele for female abdominal bristle number and sternopleural bristle number in both sexes, and an H mutant allele for both bristle traits in males and females. These results are consistent with the hypothesis that naturally occurring alleles at Dl and H contribute to quantitative genetic variation in sensory bristle number.
Salmonella Virulence Plasmid: Modular Acquisition of the spv Virulence Region by an F-Plasmid in Salmonella enterica Subspecies I and Insertion Into the Chromosome of Subspecies II, IIIa, IV and VII Isolates
Authors: Boyd E Fidelma , Hartl Daniel L
Published: April 2021

The spv operon is common to all Salmonella virulence plasmids. DNA hybridization analysis indicates that the spv region is limited in distribution to serovars of Salmonella enterica subspecies I, II, IIIa, IV, and VII and is absent from Salmonella bongori isolates. Among strains of subspecies II, IIIa, and VII, all isolates examined contained sequences that hybridized with the spv region. However, among isolates of subspecies I, DNA sequences capable of hybridizing with the spv region were found in some isolates of certain serovars. Furthermore, in isolates of subspecies I, the virulence plasmid was found in the same set of isolates as an F-related plasmid, as determined by the presence of the spv region of the virulence plasmid and the finO, traD, and repA sequences of the F-plasmid. The concordance of the virulence plasmid and all three F-plasmid sequences in subspecies I serovar Choleraesuis, Paratyphi, and Typhimurium is most easily explained if the spv region is carried in an F-related plasmid in these isolates. In contrast, among S. enterica subspecies II, IIIa, IV, and VII, the isolates that contain spv sequences did not hybridize with an F-related plasmid or any other identifiable plasmid. With the use of pulse-field gel electrophoresis, the spv region in subspecies II, IIIa, and VII was found to be encoded on the chromosome. Analysis of the phylogenetic distribution of spv among Salmonella isolates and comparative nucleotide sequence analysis of spvA and spvC suggests that the spv region was acquired very recently, after speciation of the salmonellae.
The M26 Hotspot of Schizosaccharomyces pombe Stimulates Meiotic Ectopic Recombination and Chromosomal Rearrangements
Authors: Virgin Jeffrey B , Bailey Jeffrey P
Published: April 2021

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10–1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35–60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.
On the Sampling Variance of Intraclass Correlations and Genetic Correlations
Authors: Visscher Peter M
Published: April 2021

Widely used standard expressions for the sampling variance of intraclass correlations and genetic correlation coefficients were reviewed for small and large sample sizes. For the sampling variance of the intraclass correlation, it was shown by simulation that the commonly used expression, derived using a first-order Taylor series performs better than alternative expressions found in the literature, when the between-sire degrees of freedom were small. The expressions for the sampling variance of the genetic correlation are significantly biased for small sample sizes, in particular when the population values, or their estimates, are close to zero. It was shown, both analytically and by simulation, that this is because the estimate of the sampling variance becomes very large in these cases due to very small values of the denominator of the expressions. It was concluded, therefore, that for small samples, estimates of the heritabilities and genetic correlations should not be used in the expressions for the sampling variance of the genetic correlation. It was shown analytically that in cases where the population values of the heritabilities are known, using the estimated heritabilities rather than their true values to estimate the genetic correlation results in a lower sampling variance for the genetic correlation. Therefore, for large samples, estimates of heritabilities, and not their true values, should be used.
Stochastic Kinetic Analysis of Developmental Pathway Bifurcation in Phage λ-Infected Escherichia coli Cells
Authors: Arkin Adam , Ross John , McAdams Harley H
Published: April 2021

Fluctuations in rates of gene expression can produce highly erratic time patterns of protein production in individual cells and wide diversity in instantaneous protein concentrations across cell populations. When two independently produced regulatory proteins acting at low cellular concentrations competitively control a switch point in a pathway, stochastic variations in their concentrations can produce probabilistic pathway selection, so that an initially homogeneous cell population partitions into distinct phenotypic subpopulations. Many pathogenic organisms, for example, use this mechanism to randomly switch surface features to evade host responses. This coupling between molecular-level fluctuations and macroscopic phenotype selection is analyzed using the phage λ lysis-lysogeny decision circuit as a model system. The fraction of infected cells selecting the lysogenic pathway at different phage:cell ratios, predicted using a molecular-level stochastic kinetic model of the genetic regulatory circuit, is consistent with experimental observations. The kinetic model of the decision circuit uses the stochastic formulation of chemical kinetics, stochastic mechanisms of gene expression, and a statistical-thermodynamic model of promoter regulation. Conventional deterministic kinetics cannot be used to predict statistics of regulatory systems that produce probabilistic outcomes. Rather, a stochastic kinetic analysis must be used to predict statistics of regulatory outcomes for such stochastically regulated systems.
Analysis of fluG Mutations That Affect Light-Dependent Conidiation in Aspergillus nidulans
Authors: Yager Lawrence N , Lee Hyung-Ok , Nagle Deborah L , Zimmerman John E
Published: April 2021

Conidiation in Aspergillus nidulans is induced by exposure to red light but can also be induced by blue light in certain mutant strains. We have isolated a mutation in the fluG gene that abolishes responsiveness to red light but does not affect the response to blue light. It has been shown that the veA1 (velvet) mutation allows conidiation to occur in the absence of light. We have identified three other fluG mutations that suppress the veA1 phenotype; these double mutants do not conidiate in the dark. The mutations described here define two new phenotypic classes of fluG alleles that display abnormal responses to light. We have characterized these mutations with respect to their molecular identity and to their effect on fluG transcription. Although it has been shown that fluG is required for the synthesis of an extracellular factor that directs conidiation, we do not detect this factor under conditions that promote conidiation in the veA1 suppressors. Furthermore, extracellular rescue is not observed in fluG deletion strains containing the wild-type veA allele. We propose that a genetic interaction between fluG and veA influences the production of the extracellular signal and regulates the initiation of conidiation.
Gain-of-Function Mutations in the Caenorhabditis elegans lin-1 ETS Gene Identify a C-Terminal Regulatory Domain Phosphorylated by ERK MAP Kinase
Authors: Jacobs Dave , Beitel Greg J , Clark Scott G , Horvitz H Robert , Kornfeld Kerry
Published: April 2021

Genetic analysis of lin-1 loss-of-function mutations suggests that lin-1 controls multiple cell-fate decisions during Caenorhabditis elegans development and is negatively regulated by a conserved receptor tyrosine kinase-Ras-ERK mitogen-activated protein (MAP) kinase signal transduction pathway. LIN-1 protein contains an ETS domain and presumably regulates transcription. We identified and characterized six gain-of-function mutations that define a new class of lin-1 allele. These lin-1 alleles appeared to be constitutively active and unresponsive to negative regulation. Each allele has a single-base change that affects the predicted C terminus of LIN-1, suggesting this region is required for negative regulation. The C terminus of LIN-1 was a high-affinity substrate for Erk2 in vitro, suggesting that LIN-1 is directly regulated by ERK MAP kinase. Because mpk-1 ERK MAP kinase controls at least one cell-fate decision that does not require lin-1, our results suggest that MPK-1 contributes to the specificity of this receptor tyrosine kinase-Ras-MAP kinase signal transduction pathway by phosphorylating different proteins in different developmental contexts. These lin-1 mutations all affect a four-amino-acid motif, FQFP, that is conserved in vertebrate and Drosophila ETS proteins that are also phosphorylated by ERK MAP kinase. This sequence may be a substrate recognition motif for the ERK subfamily of MAP kinases.
Molecular Population Genetics of the Southern Elephant Seal Mirounga leonina
Authors: Slade Robert W , Moritz Craig , Hoelzel A Rus , Burton Harry R
Published: April 2021

Southern elephant seals breed on sub-Antarctic islands and have a circumpolar distribution. We assayed mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) variation in the three main populations in the south Atlantic, south Indian, and south Pacific oceans, and a smaller continental population in South America. Population structure of mtDNA was strong and not consistent with isolation by distance. The nDNA loci, although less informative, were consistent with the mtDNA results. Geographic structure appears to be dominated by historical processes, not contemporary gene flow. Uncorrected levels of nucleotide diversity for mtDNA control region I (2.86%) and nDNA (0.09%) were similar to those in humans and mice. Mutation rates for control region I (75 × 10−9 substitutions per site per year) and nDNA (1.23 × 10−9) were similar to those in other mammals. Female effective population size and total effective population size were roughly equal at ~4 × 104, indicating a twofold greater rate of drift for mtDNA. Effective breeding sex ratio of four to five females per male was estimated from nucleotide diversity and mutation rates for mtDNA and nDNA, and was much less than behavioral observations would suggest. There was no evidence for selection at any of the assayed loci.
Identification of a Calcineurin-Independent Pathway Required for Sodium Ion Stress Response in Saccharomyces cerevisiae
Authors: Ganster Raymond W , McCartney Rhonda R , Schmidt Martin C
Published: April 2021

The calcium-dependent protein phosphatase calcineurin plays an essential role in ion homeostasis in yeast. In this study, we identify a parallel ion stress response pathway that is independent of the calcineurin signaling pathway. Cells with null alleles in both STD1 and its homologue, MTH1, manifest numerous phenotypes observed in calcineurin mutants, including sodium, lithium, manganese, and hydroxyl ion sensitivity, as well as alpha factor toxicity. Furthermore, increased gene dosage of STD1 suppresses the ion stress phenotypes in calcineurin mutants and confers halotolerance in wild-type cells. However, Std1p functions in a calcineurin-independent ion stress response pathway, since a std1 mth1 mutant is FK506 sensitive under conditions of ion stress. Mutations in other genes known to regulate gene expression in response to changes in glucose concentration, including SNF3, RGT2, and SNF5, also affect cell growth under ion stress conditions. Gene expression studies indicate that the regulation of HAL1 and PMR2 expression is affected by STD1 gene dosage. Taken together, our data demonstrate that response to ion stress requires the participation of both calcineurin-dependent and -independent pathways.
Flowering-Time Genes Modulate the Response to LEAFY Activity
Authors: Nilsson Ove , Lee Ilha , Blázquez Miguel A , Weigel Detlef
Published: April 2021

Among the genes that control the transition to flowering in Arabidopsis is a large group whose inactivation causes a delay in flowering. It has been difficult to establish different pathways in which the flowering-time genes might act, because mutants with lesions in these genes have very similar phenotypes. Among the putative targets of the flowering-time genes is another group of genes, which control the identity of individual meristems. Overexpression of one of the meristem-identity genes, LEAFY, can cause the precocious generation of flowers and thus early flowering. We have exploited the opposite phenotypes seen in late-flowering mutants and LEAFY overexpressers to clarify the genetic interactions between flowering-time genes and LEAFY. According to epistatic relationships, we can define one class of flowering-time genes that affects primarily the response to LEAFY activity, and another class of genes that affects primarily the transcriptional induction of LEAFY. These observations allow us to expand previously proposed models for the genetic control of flowering time.