bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature-responsive structural reversibility of FGF21 and structure-based design of its variant with enhanced potency
Authors: Jung Ye-Eun , Lee Kyeong Won , Cho Jae Hyun , Bae Da-Woon , Jeong Bo-Gyeong , Jung Yeon-Ju , An Young Jun , Kim Kyungchan , Lee Ga Seul , Kang Lin-Woo , Moon Jeong Hee , Lee Jung-Hyun , Kim Eun-Kyoung , Yim Hyung-Soon , Cha Sun-Shin
Published: November 2021

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between β10 and β12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to in vitro cell-based assays and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.
Improving Peptide-Protein Docking with AlphaFold-Multimer using Forced Sampling
Authors: Johansson-Åkhe Isak , Wallner Björn
Published: November 2021

Protein interactions are key in vital biological process. In many cases, particularly often in regulation, this interaction is between a protein and a shorter peptide fragment. Such peptides are often part of larger disordered regions of other proteins. The flexible nature of peptides enable rapid, yet specific, regulation of important functions in the cell, such as the cell life-cycle. Because of this, understanding the molecular details of these interactions are crucial to understand and alter their function, and many specialized computational methods have been developed to study them.

The recent release of AlphaFold and AlphaFold-Multimer has caused a leap in accuracy for computational modeling of proteins. In this study, the ability of AlphaFold to predict which peptides and proteins interact as well as its accuracy in modeling the resulting interaction complexes are benchmarked against established methods in the fields of peptide-protein interaction prediction and modeling. We find that AlphaFold-Multimer consistently produces predicted interaction complexes with a median DockQ of 0.47 for all 112 complexes investigated. Additionally, it can be used to separate interacting from non-interacting pairs of peptides and proteins with ROC-AUC and PR-AUC of 0.78 and 0.61, respectively, best among the method benchmarked.

However, the most interestingly result is the possibility to improve AlphaFold by enabling dropout at inference to sample a wider part of the conformational space. This improves the median DockQ from 0.47 to 0.56 for rank 1 and the median best DockQ improves from 0.58 to 0.72. This scheme of generating more structures with AlphaFold should be generally useful for many application involving multiple states, flexible regions and disorder.
Population dynamics underlying associative learning in the dorsal and ventral hippocampus
Authors: Biane Jeremy S. , Ladow Max A. , Stefanini Fabio , Boddu Sayi P. , Fan Austin , Hassan Shazreh , Dundar Naz , Apodaca-Montano Daniel L. , Woods Nicholas I. , Kheirbek Mazen A.
Published: November 2021

Animals associate cues with outcomes and continually update these associations as new information is presented. The hippocampus is crucial for this, yet how neurons track changes in cue-outcome associations remains unclear. Using 2-photon calcium imaging, we tracked the same dCA1 and vCA1 neurons across days to determine how responses evolve across phases of odor-outcome learning. We find that, initially, odors elicited robust responses in dCA1, whereas in vCA1 responses emerged after learning, including broad representations that stretched across cue, trace, and outcome periods. Population dynamics in both regions rapidly reorganized with learning, then stabilized into ensembles that stored odor representations for days, even after extinction or pairing with a different outcome. Finally, we found stable, robust signals across CA1 when anticipating reward, but not when anticipating inescapable shock. These results identify how the hippocampus encodes, stores, and updates learned associations, and illuminates the unique contributions of dorsal and ventral hippocampus.
Brain state and cortical layer-specific mechanisms underlying perception at threshold
Authors: Morton Mitchell P , Denagamage Sachira , Blume Isabel J , Reynolds John H , Jadi Monika P , Nandy Anirvan S
Published: November 2021

Identical stimuli can be perceived or go unnoticed across successive presentations, producing divergent behavioral outcomes despite similarities in sensory input. We sought to understand how fluctuations in behavioral state and cortical layer and cell-class specific neural activity underlie this perceptual variability. We analyzed physiological measurements of state and laminar electrophysiological activity in visual area V4 while monkeys were rewarded for correctly reporting a stimulus change at perceptual threshold. Hit trials were characterized by a behavioral state with heightened arousal, greater eye position stability, and enhanced decoding performance of stimulus identity from neural activity. Target stimuli evoked stronger responses in V4 in hit trials, and excitatory neurons in the superficial layers, the primary feed-forward output of the cortical column, exhibited lower variability. Feed-forward interlaminar population correlations were stronger in hits. Hit trials were further characterized by greater synchrony between the output layers of the cortex during spontaneous activity, while the stimulus-evoked period showed elevated synchrony in the feed-forward pathway. Taken together, these results suggest that a state of elevated arousal and stable retinal images allow enhanced processing of sensory stimuli, which contributes to hits at perceptual threshold.
Sleep loss disrupts the neural signature of successful learning
Authors: Guttesen Anna á V. , Gaskell M. Gareth , Madden Emily V. , Appleby Gabrielle , Cross Zachariah R. , Cairney Scott A.
Published: November 2021

Sleep supports memory consolidation as well as next-day learning. The influential Active Systems account of offline consolidation suggests that sleep-associated memory processing paves the way for new learning, but empirical evidence in support of this idea is scarce. Using a within-subjects (N = 30), crossover design, we assessed behavioural and electrophysiological indices of episodic encoding after a night of sleep or total sleep deprivation in healthy adults (aged 18-25 years), and investigated whether behavioural performance was predicted by the overnight consolidation of episodic associations formed the previous day. Sleep supported memory consolidation and next-day learning, as compared to sleep deprivation. However, the magnitude of this sleep-associated consolidation benefit did not significantly predict the ability to form novel memories after sleep. Interestingly, sleep deprivation prompted a qualitative change in the neural signature of encoding: whereas 12-20 Hz beta desynchronization – an established marker of successful encoding – was observed after sleep, sleep deprivation disrupted beta desynchrony during successful learning. Taken together, these findings suggest that effective learning depends on sleep, but not necessarily sleep-associated consolidation.
Dithiothreitol causes toxicity in C. elegans by modulating the methionine-homocysteine cycle
Authors: G Gokul , Singh Jogender
Published: November 2021

The redox reagent dithiothreitol (DTT) causes stress in the endoplasmic reticulum (ER) by disrupting its oxidative protein folding environment, which results in the accumulation and misfolding of the newly synthesized proteins. DTT may potentially impact cellular physiology by ER-independent mechanisms; however, such mechanisms remain poorly characterized. Using the nematode model Caenorhabditis elegans, here we show that DTT toxicity is modulated by the bacterial diet. Specifically, the dietary component vitamin B12 alleviates DTT toxicity in a methionine synthase-dependent manner. Using a forward genetic screen, we discover that loss-of-function of R08E5.3, an S-adenosylmethionine (SAM)-dependent methyltransferase, confers DTT resistance. DTT upregulates R08E5.3 expression and modulates the activity of the methionine-homocysteine cycle. Employing genetic studies, we establish that DTT toxicity is a result of the depletion of SAM. Finally, we show that a functional IRE-1/XBP-1 unfolded protein response pathway is required to counteract toxicity at high, but not low, DTT concentrations.
Identification of shared bacterial strains in the vaginal microbiota of reproductive-age mothers and daughters using genome-resolved metagenomics
Authors: France M. , Brown S. , Rompalo A. , Brotman R. M. , Ravel J.
Published: November 2021

It has been suggested that the human microbiome might be vertically transmitted from mother to offspring and that early colonizers may play a critical role in development of the immune system. Studies have shown limited support for the vertical transmission of the intestinal microbiota but the derivation of the vaginal microbiota remains largely unknown. Although the vaginal microbiota of children and reproductive age cis women differ in composition, the vaginal microbiota could be vertically transmitted. To determine whether there was any support for this hypothesis, we examined the vaginal microbiota of daughter-mother pairs from the Baltimore metropolitan area (ages 14-27, 32-51; n=39). We assessed whether the daughter’s microbiota was similar in composition to their mother’s using metataxonomics. Permutation tests revealed that while some pairs did have similar vaginal microbiota, the degree of similarity did not exceed that expected by chance. Genome-resolved metagenomics was used to identify shared bacterial strains in a subset of the families (n=22). We found a small number of bacterial strains that were shared between mother-daughter pairs but identified more shared strains between individuals from different families, indicating that vaginal bacteria may display biogeographic patterns. Earlier-in-life studies are needed to demonstrate vertical transmission of the vaginal microbiota.

Importance

Early colonizers of our microbiota are theorized to play an important role in the development of our immune system, yet we know little about how these communities are established. Vertical transmission from mother to offspring at the time of birth is theorized to be a major source of early colonizers but limited evidence supporting this process has only been shown for the intestinal tract microbiota. The provenance of the vaginal microbiota is largely unknown, although some have posited it is similarly vertically transmitted. We examined the vaginal microbiota of mother-daughter pairs and found limited evidence in support of this hypothesis. However, our analysis also revealed putative biogeographic patterns in the distribution of the strains which comprise the vaginal microbiota. Our results give insight into the role of vertical transmission for the vaginal microbiota and motivate future studies on the biogeography of these bacteria.
Expression of the cyanobacterial F0F1 ATP synthase inhibitor AtpΘ depends on small basic DNA-binding proteins and differential mRNA stability
Authors: Song Kuo , Hagemann Martin , Georg Jens , Maaß Sandra , Becher Dörte , Hess Wolfgang R.
Published: November 2021

F0F1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. AtpΘ is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. AtpΘ inhibits the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, AtpΘ is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5’UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2 and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that basic transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status.

IMPORTANCE

F0F1 ATP synthases are protein complexes that produce ATP, the universal biological energy source in all kinds of organisms. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis but they cannot use the same mechanism as plants that inhibit chloroplast ATP synthases entirely during the night because respiratory and photosynthetic complexes are both located in the same membrane system. AtpΘ is a small peptide inhibitor of the reverse ATPase function in cyanobacteria encoded by the gene atpT. The production of AtpΘ is highly regulated to ensure that it is only synthetized when it is needed. In the here presented work we found that three transcription factors contribute to the regulation of atpT expression. However, we identified the control of mRNA stability as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the AtpΘ-based inhibitory mechanism within the context of the cellular redox and energy balance.
Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos
Authors: Cunningham Neil H. J. , Bouhlel Imène B. , Conduit Paul T.
Published: November 2021

Centrosomes are important organisers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material (PCM), which nucleates and organises the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new “daughter” centriole is built orthogonally on one side of each radially symmetric “mother” centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.
The transcription factor AtHB23 modulates starch turnover for root development and plant survival under salinity
Authors: Perotti María Florencia , Arce Agustín Lucas , Ariel Federico Damián , Figueroa Carlos María , Chan Raquel Lía
Published: November 2021

AtHB23 is a homeodomain-leucine zipper I transcription factor, previously characterized as a modulator of lateral root initiation and higher-order roots development. The role of this gene in response to salinity stress was completely unknown. To elucidate the role of AtHB23 in response to salinity stress, we combined histochemical β-glucuronidase (GUS) analysis, root phenotyping, starch staining, optic and electronic transmission microscopy, expression studies by RT-qPCR, and transcriptome analysis of silenced, overexpressor, and crossed plants. We revealed that the expression pattern of AtHB23 is regulated by NaCl in the main and lateral roots, affecting the root phenotype. A severe reduction in primary root length, a significant increment in the initiation of lateral roots, and a low survival rate in salinity conditions were observed in AtHB23-silenced plants, whereas AtHB23 overexpressors showed the opposite phenotype. These developmental defects were explained by the degradation of starch granules and an alteration in starch metabolism. The AtHB23-target gene LAX3 is repressed in the tip of the main root and affected by NaCl.

We conclude that AtHB23 is vital for plant survival and adaptation to salt stress conditions, and its function is related to the gravitropic response mediated by starch granule turnover, involving the auxin carrier LAX3.

Highlight

The transcription factor AtHB23 is crucial for plant survival and adaptation to salt stress conditions, and its function is related to the gravitropic response mediated by starch-granule turnover, involving LAX3.
The natural frequencies of the resting human brain: an MEG-based atlas
Authors: Capilla Almudena , Arana Lydia , García-Huéscar Marta , Melcón María , Gross Joachim , Campo Pablo
Published: November 2021

Brain oscillations are considered to play a pivotal role in neural communication. However, detailed information regarding the typical oscillatory patterns of individual brain regions is surprisingly scarce. In this study we applied a multivariate data-driven approach to create an atlas of the natural frequencies of the resting human brain on a voxel-by-voxel basis. We analysed resting-state magnetoencephalography (MEG) data from 128 healthy adult volunteers obtained from the Open MEG Archive (OMEGA). Spectral power was computed in source space in 500 ms steps for 82 frequency bins logarithmically spaced from 1.7 to 99.5 Hz. We then applied k-means clustering to detect characteristic spectral profiles and to eventually identify the natural frequency of each voxel. Our results revealed a region-specific organisation of intrinsic oscillatory activity, following both a medial-to-lateral and a posterior-to-anterior gradient of increasing frequency. In particular, medial fronto-temporal regions were characterised by slow rhythms (delta/theta). Posterior regions presented natural frequencies in the alpha band, although with differentiated generators in the precuneus and in sensory-specific cortices (i.e., visual and auditory). Somatomotor regions were distinguished by the mu rhythm, while the lateral prefrontal cortex was characterised by oscillations in the high beta range (>20 Hz). Importantly, the brain map of natural frequencies was highly replicable in two independent subsamples of individuals. To the best of our knowledge, this is the most comprehensive atlas of ongoing oscillatory activity performed to date. Furthermore, the identification of natural frequencies is a fundamental step towards a better understanding of the functional architecture of the human brain.
The mouse mammary tumor virus intasome exhibits distinct dynamics on target DNA
Authors: Baltierra-Jasso Laura E. , Jones Nathan D. , Ballandras-Colas Allison , Engelman Alan N. , Fishel Richard , Yoder Kristine E.
Published: November 2021

Retroviral intasomes are complexes assembled from purified integrase (IN) and oligonucleotides mimicking viral DNA ends (vDNA). Recombinant intasomes faithfully recapitulate integration of vDNA into a target DNA. Structural studies of retroviral intasomes have revealed an array of IN oligomer forms, which appear to share a conserved intasome core coordinating the vDNA ends for strand transfer into the target DNA. Here we have explored the biochemical and dynamic properties of the mouse mammary tumor virus (MMTV) octameric intasome. We show that the MMTV intasome is remarkably stable compared to the prototype foamy virus (PFV) tetrameric intasome. MMTV integration activity peaks within the range of physiological ionic strength and is more active in the presence of manganese compared to magnesium. Single-molecule images demonstrate that the target DNA search by MMTV intasomes appears rate-limiting, similar to PFV intasomes. The time between strand transfer of the two MMTV vDNA ends into the target DNA is ∼3 fold slower than PFV intasomes. MMTV intasomes can form extremely stable, largely immobile filaments on a target DNA that are comprised of multiple intasomes. This unusual property suggests that MMTV intasomes may readily form higher order oligomers that might underpin their increased stability.
Myogenetic oligodeoxynucleotide restores differentiation and reverses inflammation of myoblasts aggravated by cancer-conditioned medium
Authors: Nihashi Yuma , Yamamoto Machi , Shimosato Takeshi , Takaya Tomohide
Published: November 2021

Cancer cachexia is characterized by irreversible muscle loss which is a critical factor in the prognosis of cancer patients. Myoblasts are myogenic precursor cells that are required to maintain skeletal muscle tissue. Previous studies have reported that cancer-released factors deteriorate myoblast differentiation, which is one of the causes of cachexia-associated muscle wasting. We recently identified the myogenetic oligodeoxynucleotide iSN04, which acts an anti-nucleolin aptamer and promotes myogenesis. The present study investigated the effects of iSN04 on human myoblasts exposed to conditioned medium (CM) of colon cancer cells. Cancer-CM impaired myogenic differentiation and myotube formation of myoblasts by upregulating the expression of inflammatory cytokines. iSN04 completely reversed cancer-CM-induced deteriorated myogenesis and inflammatory responses in myoblasts. Tumor necrosis factor-α (TNF-α), a representative cytokine present in cancer-CM, inhibited differentiation and induced inflammation of myoblasts, similar to cancer-CM. Pre-treatment with iSN04 reversed TNF-α-induced cachectic phenotypic features in myoblasts. These results indicate that iSN04 protects myoblasts against the effects of cancer-released factors and maintain their myogenic activity. This study provides a novel therapeutic strategy to prevent muscle loss associated with cancer cachexia.
Mechanical torque promotes bipolarity of the mitotic spindle through multi-centrosomal clustering
Authors: Miles Christopher E. , Zhu Jie , Mogilner Alex
Published: November 2021

Intracellular forces shape cellular organization and function. One example is the mi-totic spindle, a cellular machine consisting of multiple chromosomes and centrosomes which interact via dynamic microtubule filaments and motor proteins, resulting in complicated spatially dependent forces. For a cell to divide properly, is important for the spindle to be bipolar, with chromosomes at the center and multiple centrosomes clustered into two ‘poles’ at opposite sides of the chromosomes. Experimental observations show that in unhealthy cells, the spindle can take on a variety of patterns. What forces drive each of these patterns? It is known that attraction between centrosomes is key to bipolarity, but what the prevents the centrosomes from collapsing into a monopolar configuration? Here, we explore the hypothesis that torque rotating chromosome arms into orientations perpendicular to the centrosome-centromere vector promotes spindle bipolarity. To test this hypothesis, we construct a pairwise-interaction model of the spindle. On a continuum version of the model, an integro-PDE system, we perform linear stability analysis and construct numerical solutions which display a variety of spatial patterns. We also simulate a discrete particle model resulting in a phase diagram that confirms that the spindle bipolarity emerges most robustly with torque. Altogether, our results suggest that rotational forces may play an important role in dictating spindle patterning.
Impact of pre-anthesis drought stress on physiology, yield-related traits and drought responsive genes in green super rice
Authors: Ahmad Hassaan , Zafar Syed Adeel , Naeem Muhammad Kashif , Shokat Sajid , Inam Safeena , Naveed Amir Shahzad , Xu Jianlong , Li Zhikhang , Ali Ghulam Muhammad , Khan Muhammad Ramzan
Published: November 2021

Optimum soil water availability is vital for maximum yield production in rice which is challenged by increasing spells of drought. The reproductive stage drought is among the main limiting factors leading to the drastic reduction in grain yield. Objective of this study was to investigate the molecular and morpho-physiological responses of pre-anthesis stage drought stress in green super rice. The study assessed the performance of 26 rice lines under irrigated and drought conditions. Irrigated treatment was allowed to grow normally while drought stress was imposed for 30 days at pre-anthesis stage. Three important physiological traits including pollen fertility percentage (PFP), cell membrane stability (CMS) and normalized difference vegetative index (NDVI) were recorded at anthesis stage during the last week of drought stress. Agronomic traits of economic importance including grain yield were recorded at maturity stage. The analysis of variance demonstrated significant variation among the genotypes for most of the studied traits. Correlation and principal component analyses demonstrated highly significant associations of particular agronomic traits with grain yield, and genetic diversity among genotypes, respectively. Our study demonstrated a higher drought tolerance potential of GSR lines compared to local cultivars, mainly by higher pollen viability, plant biomass, CMS, and harvest index under drought. In addition, the molecular basis of drought tolerance in GSR lines was related to upregulation of certain drought responsive genes includingOsSADRI, OsDSM1, andOsDT11. Our study identified novel drought tolerance related genes (OsDRG-1, OsDRG-2, OsDRG-3andOsDRG-4)that could be further characterized using reverse genetics to be utilized in molecular breeding for drought tolerance.
Decay Rates of Human mRNAs: Correlation With Functional Characteristics and Sequence Attributes
Authors: Yang Edward , van Nimwegen Erik , Zavolan Mihaela , Rajewsky Nikolaus , Schroeder Mark , Magnasco Marcelo , Darnell James E.
Published: November 2021

Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions. We decided to measure mRNA decay rates in two human cell lines with high-density oligonucleotide arrays that enable the measurement of decay rates simultaneously for thousands of mRNA species. Using existing annotation and the Gene Ontology hierarchy of biological processes, we assign mRNAs to functional classes at various levels of resolution and compare the decay rate statistics between these classes. The results show statistically significant organizational principles in the variation of decay rates among functional classes. In particular, transcription factor mRNAs have increased average decay rates compared with other transcripts and are enriched in “fast-decaying” mRNAs with half-lives <2 h. In contrast, we find that mRNAs for biosynthetic proteins have decreased average decay rates and are deficient in fast-decaying mRNAs. Our analysis of data from a previously published study of Saccharomyces cerevisiae mRNA decay shows the same functional organization of decay rates, implying that it is a general organizational scheme for eukaryotes. Additionally, we investigated the dependence of decay rates on sequence composition, that is, the presence or absence of short mRNA motifs in various regions of the mRNA transcript. Our analysis recovers the positive correlation of mRNA decay with known AU-rich mRNA motifs, but we also uncover further short mRNA motifs that show statistically significant correlation with decay. However, we also note that none of these motifs are strong predictors of mRNA decay rate, indicating that the regulation of mRNA decay is more complex and may involve the cooperative binding of several RNA-binding proteins at different sites.
Expanding the Use of Zymography by the Chemical Linkage of Small, Defined Substrates to the Gel Matrix
Authors: Kaberdin Vladimir R. , McDowall Kenneth J.
Published: November 2021

In the postgenomic era, the comprehensive proteomic analysis of metabolic and signaling pathways is inevitably faced with the challenge of large-scale identification and characterization of polypeptides with a particular enzymatic activity. Previous work has shown that a wide variety of enzymatic activities of microbial, plant, and animal origin can be assigned to individual polypeptides using in-gel activity staining (zymography). However, a number of limitations, such as special substrate requirements, the lack of a standard procedure, and difficulties in distinguishing enzymes with overlapping activities have precluded the widespread use of zymography as a routine laboratory method. Here we demonstrate that, by employing small-defined substrates that are covalently attached to the gel matrix, we can largely overcome the aforementioned problems and assay readily a number of different classes of enzymatic activities within gels after standard SDS-polyacrylamide electrophoresis. Moreover, this development is compatible with the two-dimensional separation of proteins and thus has great potential in the high-throughput screening and characterization of complex biological and clinical samples.
Detection of Deleted Genomic DNA Using a Semiautomated Computational Analysis of GeneChip Data
Authors: Salamon Hugh , Kato-Maeda Midori , Small Peter M. , Drenkow Jorg , Gingeras Thomas R.
Published: November 2021

Genomic diversity within and between populations is caused by single nucleotide mutations, changes in repetitive DNA systems, recombination mechanisms, and insertion and deletion events. The contribution of these sources to diversity, whether purely genetic or of phenotypic consequence, can only be investigated if we have the means to quantitate and characterize diversity in many samples. With the advent of complete sequence characterization of representative genomes of different species, the possibility of developing protocols to screen for genetic polymorphism across entire genomes is actively being pursued. The large numbers of measurements such approaches yield demand that we pay careful attention to the numerical analysis of data. In this paper we present a novel application of an Affymetrix GeneChip to perform genome-wide screens for deletion polymorphism. A high-density oligonucleotide array formatted for mRNA expression and targeted at a fully sequenced 4.4-million–base pair Mycobacterium tuberculosis standard strain genome was adapted to compare genomic DNA. Hybridization intensities to 111,000 probe pairs (perfect complement and mismatch complement) were measured for genomic DNA from a clinical strain and from a vaccine organism. Because individual probe-pair hybridization intensities exhibit limited sensitivity/specificity characteristics to detect deletions, data-analytical methodology to exploit measurements from multiple probes in tandem locations across the genome was developed. The TSTEP (Tandem Set Terminal Extreme Probability) algorithm designed specifically to analyze the tandem hybridization measurements data was applied and shown to discover genomic deletions with high sensitivity. The TSTEP algorithm provides a foundation for similar efforts to characterize deletions in many hybridization measures in similar-sized and larger genomes. Issues relating to the design of genome content screening experiments and the implications of these methods for studying population genomics and the evolution of genomes are discussed.
Assessment of Compositional Heterogeneity Within and Between Eukaryotic Genomes
Authors: Nekrutenko Anton , Li Wen-Hsiung
Published: November 2021

Using large amounts of long genomic sequences, we studied the compositional patterns of eukaryotic genomes. We developed a simple measure, the compositional heterogeneity (or variability) index, to compare the differences in compositional heterogeneity between long genomic sequences. The index measures the average difference in GC content between two adjacent windows normalized by the standard error expected under the assumption of random distribution of nucleotides in a window. We report the following findings: (1) The extent of the compositional heterogeneity in a genomic sequence strongly correlates with its GC content in all multicellular eukaryotes studied regardless of genome size. (2) The human genome appears to be highly compositionally heterogeneous both within and between individual chromosomes; the heterogeneity goes much beyond the predictions of the isochore model. (3) All genomes of multicellular eukaryotes examined in this study are compositionally heterogeneous, although they also contain compositionally uniform segments, or isochores. (4) The true uniqueness of the human (or mammalian) genome is the presence of very high GC regions, which exhibit unusually high compositional heterogeneity and contain few long homogeneous segments (isochores). In general, GC-poor isochores tend to be longer than GC-rich ones. These findings indicate that the genomes of multicellular organisms are much more heterogeneous in nucleotide composition than depicted by the isochore model and so lead to a looser definition of isochores.
Mutation Detection Using Mass Spectrometric Separation of Tiny Oligonucleotide Fragments
Authors: Elso Colleen , Toohey Brendan , Reid Gavin E. , Poetter Karl , Simpson Richard J. , Foote Simon J.
Published: November 2021

A DNA mutation detection protocol able to identify and characterize a previously unknown change in a given sequence in a rapid, efficient, sensitive, and inexpensive manner is required to take advantage of the resources now available to researchers through the genome sequencing projects. We have developed a method based on base-specific cleavage of polymerase chain reaction (PCR) products and then separation of the fragments by matrix-assisted laser desorption ionization–mass spectrometry (MALDI-MS), which can meet these criteria. Differences are seen as the presence, absence, or mass change of peaks corresponding to fragments affected by the base difference. This technique is shown through the detection of a polymorphism in the 3′ untranslated region of IL12p40 from a double-stranded PCR product, and the detection of a single nucleotide polymorphism between two mouse strains. The sensitivity of the technique can be increased with the use of postsource decay, which enables differentiation of two fragments of identical mass but different sequence. The level of specificity and the rapid sample analysis time lend this technique to the mass screening of individuals for sequence changes and, in combination with MS sequencing methods, could be used to facilitate rapid resequencing of DNA.
Gene Discovery Using Computational and Microarray Analysis of Transcription in the Drosophila melanogaster Testis
Authors: Andrews Justen , Bouffard Gerard G. , Cheadle Chris , Lü Jining , Becker Kevin G. , Oliver Brian
Published: November 2021

Identification and annotation of all the genes in the sequencedDrosophila genome is a work in progress. Wild-type testis function requires many genes and is thus of potentially high value for the identification of transcription units. We therefore undertook a survey of the repertoire of genes expressed in the Drosophilatestis by computational and microarray analysis. We generated 3141 high-quality testis expressed sequence tags (ESTs). Testis ESTs computationally collapsed into 1560 cDNA set used for further analysis. Of those, 11% correspond to named genes, and 33% provide biological evidence for a predicted gene. A surprising 47% fail to align with existing ESTs and 16% with predicted genes in the current genome release. EST frequency and microarray expression profiles indicate that the testis mRNA population is highly complex and shows an extended range of transcript abundance. Furthermore, >80% of the genes expressed in the testis showed onefold overexpression relative to ovaries, or gonadectomized flies. Additionally, >3% showed more than threefold overexpression at p <0.05. Surprisingly, 22% of the genes most highly overexpressed in testis matchDrosophila genomic sequence, but not predicted genes. These data strongly support the idea that sequencing additional cDNA libraries from defined tissues, such as testis, will be important tools for refined annotation of the Drosophila genome. Additionally, these data suggest that the number of genes in Drosophila will significantly exceed the conservative estimate of 13,601.

[The sequence data described in this paper have been submitted to the dbEST data library under accession nos.AI944400–AI947263 and BE661985–BE662262.]

[The microarray data described in this paper have been submitted to the GEO data library under accession nos. GPLS, GSM3–GSM10.]
Ancient Genome Duplications Did Not Structure the HumanHox-Bearing Chromosomes
Authors: Hughes Austin L. , da Silva Jack , Friedman Robert
Published: November 2021

The fact that there are four homeobox (Hox) clusters in most vertebrates but only one in invertebrates is often cited as evidence for the hypothesis that two rounds of genome duplication by polyploidization occurred early in vertebrate history. In addition, it has been observed in humans and other mammals that numerous gene families include paralogs on two or more of the fourHox-bearing chromosomes (the chromosomes bearing theHoxclusters; i.e., human chromosomes 2, 7, 12, and 17), and the existence of these paralogs has been taken as evidence that these genes were duplicated along with theHoxclusters by polyploidization. We tested this hypothesis by phylogenetic analysis of 42 gene families including members on two or more of the humanHox-bearing chromosomes. In 32 of these families there was evidence against the hypothesis that gene duplication occurred simultaneously with duplication of theHoxclusters. Phylogenies of 14 families supported the occurrence of one or more gene duplications before the origin of vertebrates, and of 15 gene duplication times estimated for gene families evolving in a clock-like manner, only six were dated to the same time period early in vertebrate history during which theHoxclusters duplicated. Furthermore, of gene families duplicated around the same time as theHoxclusters, the majority showed topologies inconsistent with their having duplicated simultaneously with theHoxclusters. The results thus indicate that ancient events of genome duplication, if they occurred at all, did not play an important role in structuring the mammalianHox-bearing chromosomes.
Distribution of Protein Folds in the Three Superkingdoms of Life
Authors: Wolf Yuri I. , Brenner Steven E. , Bash Paul A. , Koonin Eugene V.
Published: November 2021

A sensitive protein-fold recognition procedure was developed on the basis of iterative database search using the PSI-BLAST program. A collection of 1193 position-dependent weight matrices that can be used as fold identifiers was produced. In the completely sequenced genomes, folds could be automatically identified for 20%–30% of the proteins, with 3%–6% more detectable by additional analysis of conserved motifs. The distribution of the most common folds is very similar in bacteria and archaea but distinct in eukaryotes. Within the bacteria, this distribution differs between parasitic and free-living species. In all analyzed genomes, the P-loop NTPases are the most abundant fold. In bacteria and archaea, the next most common folds are ferredoxin-like domains, TIM-barrels, and methyltransferases, whereas in eukaryotes, the second to fourth places belong to protein kinases, β-propellers and TIM-barrels. The observed diversity of protein folds in different proteomes is approximately twice as high as it would be expected from a simple stochastic model describing a proteome as a finite sample from an infinite pool of proteins with an exponential distribution of the fold fractions. Distribution of the number of domains with different folds in one protein fits the geometric model, which is compatible with the evolution of multidomain proteins by random combination of domains.

[Fold predictions for proteins from 14 proteomes are available on the World Wide Web atftp://ncbi.nlm.nih.gov/pub/koonin/FOLDS/index.html. The FIDs are available by anonymous ftp at the same location.]
Comparative Sequence of Human and Mouse BAC Clones from the mnd2 Region of Chromosome 2p13
Authors: Jang Wonhee , Hua Axin , Spilson Sandra V. , Miller Webb , Roe Bruce A. , Meisler Miriam H.
Published: November 2021

The mnd2 mutation on mouse chromosome 6 produces a progressive neuromuscular disorder. To determine the gene content of the 400-kb mnd2 nonrecombinant region, we sequenced 108 kb of mouse genomic DNA and 92 kb of human genomic sequence from the corresponding region of chromosome 2p13.3. Three genes with the indicated sizes and intergenic distances were identified:D6Mm5e (⩾81 kb)–787 bp–DOK (2 kb)–845 bp–LOR2 (⩾6 kb). D6Mm5e is expressed in many tissues at very low abundance and the predicted 526-residue protein contains no known functional domains. DOK encodes the p62dok rasGAP binding protein involved in signal transduction. LOR2 encodes a novel lysyl oxidase-related protein of 757 amino acid residues. We describe a simple search protocol for identification of conserved internal exons in genomic sequence. Evolutionary conservation proved to be a useful criterion for distinguishing between authentic exons and artifactual products obtained by exon amplification, RT–PCR, and 5′ RACE. Conserved noncoding sequence elements longer than 80 bp with ⩾75% nucleotide sequence identity comprise ∼1% of the genomic sequence in this region. Comparative analysis of this human and mouse genomic DNA sequence was an efficient method for gene identification and is independent of developmental stage or quantitative level of gene expression.

[The sequence data described in this paper have been submitted to the GenBank data library under the following accession numbers: AC003061, mouse BAC clone 245c12; AC003065, human BAC clone h173(E10); AF053368, mouse Lor2 cDNA; AF084363, 108-kb contig from mouse BAC 245c12; AF084364, mouse D6Mm5ecDNA.]
Postsynaptic α-Neurotoxin Gene of the Spitting Cobra, Naja naja sputatrix: Structure, Organization, and Phylogenetic Analysis
Authors: Afifiyan Fatemeh , Armugam Arunmoziarasi , Tan Chee Hong , Gopalakrishnakone Ponnampalam , Jeyaseelan Kandiah
Published: November 2021

The venom of the spitting cobra, Naja naja sputatrixcontains highly potent α-neurotoxins (NTXs) in addition to phospholipase A2 (PLA2) and cardiotoxin (CTX). In this study, we report the complete characterization of three genes that are responsible for the synthesis of three isoforms of α-NTX in the venom of a single spitting cobra. DNA amplification by long-distance polymerase chain reaction (LD-PCR) and genome walking have provided information on the gene structure including their promoter and 5′ and 3′ UTRs. Each NTX isoform is ∼4 kb in size and contains three exons and two introns. The sequence homology among these isoforms was found to be 99%. Two possible transcription sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and −45. The promoter also contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also present. The high percentage of similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with the α-NTX and κ-NTX genes from other land snakes suggests that the NTX gene has probably evolved from a common ancestral gene.

[The genomic DNA sequences reported in this paper have been submitted to GenBank databases under accession nos. AF096999 to AF097001.]
A View of Modern Human Origins from Y Chromosome Microsatellite Variation
Authors: Seielstad Mark , Bekele Endashaw , Ibrahim Muntaser , Touré Amadou , Traoré Mamadou
Published: November 2021

The idea that all modern humans share a recent (within the last 150,000 years) African origin has been proposed and supported on the basis of three observations. Most genetic loci examined to date have (1) shown greater diversity in African populations than in others, (2) placed the first branch between African and all non-African populations in phylogenetic trees, and (3) indicated recent dates for either the molecular coalescence (with the exception of some autosomal and X-chromosomal loci) or for the time of separation between African and non-African populations. We analyze variation at 10 Y chromosome microsatellite loci that were typed in 506 males representing 49 populations and every inhabited continent and find significantly greater Y chromosome diversity in Africa than elsewhere, find the first branch in phylogenetic trees of the continental populations to fall between African and all non-African populations, and date this branching with the (δμ)2 distance measure to 5800–17,400 or 12,800–36,800 years BP depending on the mutation rate used. The magnitude of the excess Y chromosome diversity in African populations appears to result from a greater antiquity of African populations rather than a greater long-term effective population size. These observations are most consistent with a recent African origin for all modern humans.
Separation of Micronuclear DNA of Stylonychia lemnae by Pulsed-Field Electrophoresis and Identification of a DNA Molecule with a High Copy Number
Authors: Maercker Christian , Kortwig Heike , Lipps Hans J.
Published: November 2021

DNA from the hypotrichous ciliatae Stylonychia lemnae was separated by PFGE. In addition to the separation of the macronuclear DNA molecules with a size up to ∼40 kb, we were able to separate the micronuclear DNA with a size between ∼90 kb and 2 Mb. One very prominent 90-kb DNA band appeared on the pulsed-field gels. We propose that this 90-kb DNA fragment represents a linear plasmid residing in the micronucleus in a very high copy number. About 10% of the micronuclear DNA consists of the 90-kb DNA molecule. It appears in the micronucleus as well as in the macronuclear anlagen during macronuclear development but not in the mature macronucleus. Thus, the multicopy DNA is eliminated during fragmentation of the macronuclear anlagen DNA in the course of macronuclear development. Therefore, this 90-kb DNA molecule might serve as an excellent tool to study the recognition and elimination of DNA during nuclear differentiation of hypotrichous ciliates.