Switchgrass diversity analysis via exome capture sequencing
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Kaeppler, Shawn↗
SEARCH · Engineering Papers
Results for “sequence analysis”
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A homolog of an Escherichia coli phosphate-binding protein gene from Xanthomonas oryzae pv. oryzae
A Xanthomonas oryzae pv. oryzae gene with sequence similarity to an Escherichia coli phosphate-binding protein gene (phoS) produces a periplasmic protein of apparent M(r) 35,000 when expressed in E. coli. Amino terminal sequencing revealed that a signal peptide is removed during transport to the periplasm in E. coli.
Non-NASA Center↗
Performance Analysis of Direct-Sequence Code-Division Multiple-Access Communications with Asymmetric Quadrature Phase-Shift-Keying Modulation
This article considers a quaternary direct-sequence code-division multiple-access (DS-CDMA) communication system with asymmetric quadrature phase-shift-keying (AQPSK) modulation for unequal error protection (UEP) capability. Both time synchronous and asynchronous cases are investigated. An expression for the probability distribution of the multiple-access interference is derived. The exact bit-error performance and the approximate performance using a Gaussian approximation and random signature sequences are evaluated by extending the techniques used for uniform quadrature phase-shift-keying (QPSK) and binary phase-shift-keying (BPSK) DS-CDMA systems. Finally, a general system model with unequal user power and the near-far problem is considered and analyzed. The results show that, for a system with UEP capability, the less protected data bits are more sensitive to the near-far effect that occurs in a multiple-access environment than are the more protected bits.
Wang, C.-W.↗
Cheap and Fast Multiplexed Bacterial Genomic Sequencing: Enabling Comparative Genomic Analysis of Antibiotic Resistance in BWA and Hospital Acquired Infections.
Abstract not provided.
Schoeniger, Joseph S.↗
CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites
High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Here, data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. Cis SERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. Cis SERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, Cis SERS utilizes the REBASE enzyme database. Results from Cis SERSenable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3’UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. Cis SERS is a java based graphical user interface built around a perl backbone. Several of the applications of Cis SERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool Cis SERSand results from in-silico and corresponding wet-lab analyses demonstrating that Cis SERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.
59 BASIC BIOLOGICAL SCIENCES↗
Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities
The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCRAC-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-TCCRAC-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-TCCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.
59 BASIC BIOLOGICAL SCIENCES↗
A1 Signature pattern and phylogenetic analysis of full-length env genes in 20 hemophiliacs infected with Korean subclade of HIV-1 subtype B
Twenty patients with hemophilia (HP) were diagnosed with HIV-1, one to two years after exposure to domestic clotting factor IX (DCF), in 1989–90. Previous analysis of pol and vif genes confirmed that HIV-1 transmission to 20 HPs occurred through infusion of DCF. In this study, we determined full-length env gene sequences (about 2,550 bp) in 21 HPs, three plasma donors whose plasma were used in DCF production, and 45 local controls. The env gene from frozen stored sera obtained 1–3 years after diagnosis as well as from samples collected years after infection, was amplified by RT-PCR, and subjected to direct sequencing. Phylogenetic analysis revealed that all sequences were subtype B, with 109 sequences from 64 patients (20 HPs, 3 serum donors, 41 local controls, LCs) belonging to the Korean subclade (KSB) and 13 sequences from 5 patients (1HP infected outside Korea, 4 LCs) not in the KSB. Sequences of the env gene from donors O and P plus the 20 HPs comprised two subclusters within KSB. In addition, signature pattern analysis revealed signature nucleotides at 45 positions between the HPs and LCs (P < 0.05). Surprisingly, specific signature nucleotides positions were conserved 100% in clusters O and P only (at 4 and 1 positions, respectively) with none in LCs. Within both clusters, sequence identity was high. These results are consistent with our previous conclusion that these 20 HPs were infected with viruses in the DCF used for treatment. In addition, we found that there are 25 signature pattern residues in amino acids in env gene of KSB distinct from subtype B.
59 BASIC BIOLOGICAL SCIENCES↗
Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis
Rationale Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. Methods Protein biomarkers of two strains of Shiga toxin–producingEscherichia coli(STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. Results Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. Conclusions TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.
Biochemistry & Molecular Biology↗
Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5
Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.
60 APPLIED LIFE SCIENCES↗
Genetic Analysis of the Full-Length gag Gene from the Earliest Korean Subclade B of HIV-1: An Outbreak among Korean Hemophiliacs
We determined the earliest full-length HIV-1 gag gene sequences in 110 patients with HIV-1, including 20 hemophiliacs (HPs) and 90 local controls (LCs). The gag gene from stored sera was amplified using RT-PCR, and was subjected to direct sequencing. Phylogenetic analysis indicated that 94 and 16 sequences belonged to the Korean subclade of HIV-1 subtype B (KSB) and subtype B, respectively. A total of 12 signature pattern amino acids were found within the KSB, distinct from the worldwide consensus of subtype B. Within the KSB, the gag gene sequences from donors O and P and those from the 20 HPs comprised two subclusters. In particular, sequences from donor O strongly clustered with those of eight HPs. Moreover, signature pattern analysis indicated that 14 signature nucleotides were shared between the HPs and LCs within KSB (p < 0.01). Among the 14 nucleotides, positions 9 and 5 belonged to clusters O and P, respectively. In conclusion, signature pattern analysis for the gag gene revealed 12 signature pattern residues within the KSB and also confirmed the previous conclusion that the 20 HPs were infected with viruses due to incompletely inactivated clotting factor IX. This study is the first genetic analysis of the HIV-1 gag gene in Korea.
59 BASIC BIOLOGICAL SCIENCES↗
MerCat: a versatile k-mer counter and diversity estimator for database-independent property analysis obtained from metagenomic and/or metatranscriptomic sequencing data
MerCat is a parallel, highly scalable and modular property software package for robust analysis of features in next-generation sequencing data. MerCat inputs include assembled contigs and raw sequence reads from any platform resulting in feature abundance counts tables. MerCat allows for direct analysis of data properties without reference sequence database dependency commonly used by search tools such as BLAST and/or DIAMOND for compositional analysis of whole community shotgun sequencing (e.g. metagenomes and metatranscriptomes).
metabolomics↗
Interactive Analysis of Functional Residues in Protein Families
For most microbes of interest, a genome sequence is available, but the function of its proteins is not known. Instead, proteins' functions are predicted from their similarity to other protein sequences.
59 BASIC BIOLOGICAL SCIENCES↗
Breathing dynamics based parameter sensitivity analysis of hetero-polymeric DNA
We study the parameter sensitivity of hetero-polymeric DNA within the purview of DNA breathing dynamics. The degree of correlation between the mean bubble size and the model parameters is estimated for this purpose for three different DNA sequences. The analysis leads us to a better understanding of the sequence dependent nature of the breathing dynamics of hetero-polymeric DNA. Out of the 14 model parameters for DNA stability in the statistical Poland-Scheraga approach, the hydrogen bond interaction ε{sub hb}(AT) for an AT base pair and the ring factor ξ turn out to be the most sensitive parameters. In addition, the stacking interaction ε{sub st}(TA-TA) for an TA-TA nearest neighbor pair of base-pairs is found to be the most sensitive one among all stacking interactions. Moreover, we also establish that the nature of stacking interaction has a deciding effect on the DNA breathing dynamics, not the number of times a particular stacking interaction appears in a sequence. We show that the sensitivity analysis can be used as an effective measure to guide a stochastic optimization technique to find the kinetic rate constants related to the dynamics as opposed to the case where the rate constants are measured using the conventional unbiased way of optimization.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
National Prevalence of Caprine Prion Protein Genetic Variability at Codons 146, 211, and 222 in Goat Herds in the United States
Scrapie is a neurodegenerative disease that impacts sheep and goats, characterized by gradual and progressive changes in neurological function. Recent research shows that the scrapie incubation period is significantly influenced by specific variations in amino acids within the prion protein gene (PRNP). The objective of this study was to estimate the national prevalence of caprine PRNP genetic variability at codons 146, 211, and 222 in goat populations across the United States. A total of 3052 blood, ear tissue, and brain tissue samples were collected from goats from 50 states. The participating states were categorized into four Veterinary Service (VS) district regions. The samples underwent DNA extraction, and the PRNP variants corresponding to codons 146, 211, and 222 were amplified and sequenced. The analysis of PRNP variants, when compared to the PRNP reference sequence, revealed seven alleles in twelve genotypes. The homozygous 146NN, 211RR, and 222QQ alleles, which have been linked to an increased risk of scrapie, were found to be the most prevalent among all the goats. The heterozygous 222QK, 211RQ, 146SD, 146ND, and 146NS alleles and the homozygous 222KK, 146SS, and 146DD alleles, known to be associated with reduced scrapie susceptibility and a prolonged incubation period after experimental challenge, were found in 1.098% (222QK), 2.33% (211RQ), 0.58% (146SD), 3.13% (146ND), 20.68% (146NS), 0.005% (222KK), 3.31% (146SS), and 0.67% (146DD) of goats, respectively. The 222QK allele was found most frequently in goats tested from the east (VS District 1, 1.59%) and southwest (VS District 4, 1.08%) regions, whereas the 211RQ allele was found most often in goats tested from the Midwest (VS District 2, 8.03%) and east (VS District 1, 6.53%) regions. The 146NS allele was found most frequently in goats tested from the northwest (VS District 3, 29.02%) and southwest (VS District 4, 20.69%) regions. Our results showed that the prevalence of less susceptible genotypes at PRNP codon 146 may be sufficient to use genetic susceptibility testing in some herds. This may reduce the number of goats removed as part of a herd clean-up plan and may promote the selective breeding goats for less susceptible alleles in high-risk herds at the national level.
59 BASIC BIOLOGICAL SCIENCES↗
A new look at dust and clouds in the Mars atmosphere - Analysis of emission-phase-function sequences from global Viking IRTM observations
The present analysis of emission-phase function (EPF) observations from the IR thermal mapper aboard the Viking Orbiter encompasses polar latitudes, and Viking Lander sites, and spans a wide range of solar longitudes. A multiple scattering radiative transfer model which incorporates a bidirectional phase function for the surface and atmospheric scattering by dust and clouds yields surface albedos and dust and ice optical properties and optical depths for the variety of Mars conditions. It is possible to fit all analyzed EPF sequences corresponding to dust scattering with an albedo of 0.92, rather than the 0.86 given by Pollack et al. on the bases of Viking Lander observations.
Clancy, R. T.↗
DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis
The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.
59 BASIC BIOLOGICAL SCIENCES↗
Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core
The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.
Evaluation Studies↗
Comprehensive analysis of differentially expressed mRNA and circRNA in Ankylosing spondylitis patients’ platelets
Highlights: • 4996 mRNAs and 2942 circRNAs were identified differentially expressed in platelets of AS patients. • Several platelet-derived immune mediators were upregulated to regulate the interaction between platelets and immune cells. • Two downregulated circRNA were identified, and the corresponding circRNA-miRNA-mRNA regulatory network was constructed. • mRNAs and circRNAs found herein could be used as diagnostic biomarkers or new therapeutic targets. Ankylosing spondylitis (AS) is a chronic inflammatory disease significantly decreasing the quality of life. Platelets play an important and active role in the development of AS. Accumulating evidence demonstrated platelets contain diverse RNA repository inherited from megakaryocytes or microvesicles. Platelet RNAs are dynamically affected by pathological conditions and could be used as diagnostic or prognostic biomarkers. However, the role of the platelet RNAs in AS is elusive. In this study, we compared mRNA and circRNA profiles in platelets between AS patients and healthy controls using RNA sequencing and bioinformatic analysis, and found 4996 mRNAs and 2942 circRNAs were differently expressed. The significantly over-expressed mRNAs in AS patients are involved in platelet activity, gap junction, focal adhesion, rap1 and toll and Imd signaling pathway. The previous identified platelet-derived immune mediators such as P2Y1, P2Y12, PF4, GPIbα, CD40L, ICAM2, CCL5 (RANTES), TGF-β (TGF-β1 and TGF-β2) and PDGF (PDGFB and PDGFA) are also included in these over expressed mRNAs, implying these factors may trigger inflammatory cascades and promote the development of AS. Additionally, we found two down-regulated circRNA (circPTPN22 and circFCHSD2) from the intersection analyses of platelets and spinal ligament tissues of AS patients. The circRNA-miRNA-mRNA regulatory network of these two circRNAs was constructed, and the target mRNAs were enriched in Th17 cell differentiation, inflammatory bowel disease, cell adhesion molecules, cytokine-cytokine receptor interaction, Jak-STAT and Wnt signaling pathway, all these pathways participate in the bone remodeling and pro/anti-inflammatory immune regulation in AS. Then, qRT-PCR was performed to validate the expression of selected key mRNAs and circRNAs and the results demonstrated that the expression levels of P2Y12, GPIbα, circPTPN22 and circFCHSD2 were consistent with the sequencing analysis. In addition, the high expression of five predicted miRNAs interacting with circPTPN22 and circFCHSD2 were also detected in AS by qRT-PCR. Taken together, our study presents a comprehensive overview of mRNAs and circRNAs in platelets in AS patients and offers new insight into the mechanisms of platelet involving in the pathogenesis of AS. The mRNAs and circRNAs identified in this study may serve as candidates for diagnosis and targeted treatment of AS.
60 APPLIED LIFE SCIENCES↗