Identification in silico and expression analysis of a β-1-4-endoglucanase and β-galactosidase genes related to ripening in guava fruit

Background Guava fruit softening is a crucial process during ripening and this process involves a number of enzymes that modifies the cell wall. Two of the enzymes that regulate this process are (a) the β-1, 4-endoglucanase 17 (BEG) which hydrolyze β-1, 4 bonds from cellulose and hemicellulose, and (b) β-galactosidase (BGA) that hydrolyzes pectin chains. Bioinformatics and expression analysis information on these genes is limited in guava fruit. Results A fragment of a β-1, 4-endoglucanase 17 (PgE17), and another of a β-galactosidase (PgGa1) were identified. These sequences have a similarity of more than 85% with those reported in the NCBI database. In the guava genome, one homologous sequence was found for PgE17 in Chr 4 and two homologous to PgGa1: one in Chr 3 and the other one in Chr 6. Putative protein PgE17 contains part of the glyco_hydro_9 domain. Putative protein PgGa1 has a part of the glyco_hydro_35 domain. Phylogenetic analysis of PgE17 and PgGa1 revealed that both are highly conserved inside the Myrtaceae family. In silico expression analysis showed that both PgE17 and PgGa1 work in a coordinated way with other cell wall modifier enzymes. Expression of these genes was found in all the guava samples analyzed. However, the highest expression was found in the fruit in the breaking and ripe states. Conclusions A β-1, 4-endoglucanase 17, and β-galactosidase 1 sequences were identified. PgE17 and PgGa1 are expressed in all the plant tissues, and fruit ripening states. Although, the highest expression was on breaker and ripe states.

Origins and breadth of pairwise epistasis in an α-helix of β-lactamase TEM-1

Epistasis affects genome evolution together with our ability to predict individual mutation effects. The mechanistic basis of epistasis remains, however, largely unknown. To quantify and better understand interactions between fitness-affecting mutations, we focus on a 11 amino-acid α-helix of the protein β-lactamase TEM-1, and build a comprehensive library of more than 15,000 double mutants. Analysis of the growth rates of these mutants shows pervasive epistasis, which can be largely explained by a non-linear two-state model, where inactivating, destabilizing, neutral, or stabilizing mutations additively contribute to the phenotype. Hence, most epistatic interactions can be predicted by a non-linear model informed by single-point mutational measurements only. Deviations from the two-state model are consistently found for few pairs of residues, in particular when they are in contact. This result, as well as single-point mutation parameters can be quantitatively found back through direct-coupling-analysis-based statistical models inferred from homologous sequence data. Our results thus shed light on the existence and the origins of the multiple determinants of the epistatic landscape, even at the level of small structural components of a protein, and suggest that the corresponding constraints shape the entire β-lactamase family.

Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain

The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.

ncDLRES: a novel method for non-coding RNAs family prediction based on dynamic LSTM and ResNet

Background Studies have proven that the same family of non-coding RNAs (ncRNAs) have similar functions, so predicting the ncRNAs family is helpful to the research of ncRNAs functions. The existing calculation methods mainly fall into two categories: the first type is to predict ncRNAs family by learning the features of sequence or secondary structure, and the other type is to predict ncRNAs family by the alignment among homologs sequences. In the first type, some methods predict ncRNAs family by learning predicted secondary structure features. The inaccuracy of predicted secondary structure may cause the low accuracy of those methods. Different from that, ncRFP directly learning the features of ncRNA sequences to predict ncRNAs family. Although ncRFP simplifies the prediction process and improves the performance, there is room for improvement in ncRFP performance due to the incomplete features of its input data. In the secondary type, the homologous sequence alignment method can achieve the highest performance at present. However, due to the need for consensus secondary structure annotation of ncRNA sequences, and the helplessness for modeling pseudoknots, the use of the method is limited. Results In this paper, a novel method “ncDLRES”, which according to learning the sequence features, is proposed to predict the family of ncRNAs based on Dynamic LSTM (Long Short-term Memory) and ResNet (Residual Neural Network). Conclusions ncDLRES extracts the features of ncRNA sequences based on Dynamic LSTM and then classifies them by ResNet. Compared with the homologous sequence alignment method, ncDLRES reduces the data requirement and expands the application scope. By comparing with the first type of methods, the performance of ncDLRES is greatly improved.

TIAMMAt: Leveraging biodiversity to revise protein domain models, evidence from innate immunity

Sequence annotation is fundamental for studying the evolution of protein families, particularly when working with non-model species. Given the rapid, ever-increasing number of species receiving high-quality genome sequencing, accurate domain modeling that is representative of species diversity is crucial for understanding protein family sequence evolution and their inferred function(s). Here, we describe a bioinformatic tool called TIAMMAt ( Taxon-Informed Adjustment of Markov Model Attributes) which revises domain profile hidden Markov models (HMMs) by incorporating homologous domain sequences from underrepresented and non-model species. Using innate immunity pathways as a case study, we show that revising profile HMM parameters to directly account for variation in homologs among underrepresented species provides valuable insight into the evolution of protein families. Following adjustment by TIAMMAt, domain profile HMMs exhibit changes in their per-site amino acid state emission probabilities and insertion/deletion probabilities while maintaining the overall structure of the consensus sequence. Our results show that domain revision can heavily impact evolutionary interpretations for some families (i.e., NLR’s NACHT domain), whereas impact on other domains (e.g., rel homology domain and interferon regulatory factor domains) is minimal due to high levels of sequence conservation across the sampled phylogenetic depth (i.e., Metazoa). Importantly, TIAMMAt revises target domain models to reflect homologous sequence variation using the taxonomic distribution under consideration by the user. TIAMMAt’s flexibility to revise any subset of the Pfam database using a user-defined taxonomic pool will make it a valuable tool for future protein evolution studies, particularly when incorporating (or focusing) on non-model species.

First Description of a Satellite DNA in Manatees’ Centromeric Regions

Trichechus manatus and Trichechus inunguis are the two Sirenia species that occur in the Americas. Despite their increasing extinction risk, many aspects of their biology remain understudied, including the repetitive DNA fraction of their genomes. Here we used the sequenced genome of T. manatus and TAREAN to identify satellite DNAs (satDNAs) in this species. We report the first description of TMAsat, a satDNA comprising ~0.87% of the genome, with ~684bp monomers and centromeric localization. In T. inunguis, TMAsat showed similar monomer length, chromosome localization and conserved CENP-B box-like motifs as in T. manatus. We also detected this satDNA in the Dugong dugon and in the now extinct Hydrodamalis gigas genomes. The neighbor-joining tree shows that TMAsat sequences from T. manatus, T. inunguis, D. dugon, and H. gigas lack species-specific clusters, which disagrees with the predictions of concerted evolution. We detected a divergent TMAsat-like homologous sequence in elephants and hyraxes, but not in other mammals, suggesting this sequence was already present in the common ancestor of Paenungulata, and later became a satDNA in the Sirenians. This is the first description of a centromeric satDNA in manatees and will facilitate the inclusion of Sirenia in future studies of centromeres and satDNA biology.

Gene Conversion amongst Alu SINE Elements

The process of non-allelic gene conversion acts on homologous sequences during recombination, replacing parts of one with the other to make them uniform. Such concerted evolution is best described as paralogous ribosomal RNA gene unification that serves to preserve the essential house-keeping functions of the converted genes. Transposed elements (TE), especially Alu short interspersed elements (SINE) that have more than a million copies in primate genomes, are a significant source of homologous units and a verified target of gene conversion. The consequences of such a recombination-based process are diverse, including multiplications of functional TE internal binding domains and, for evolutionists, confusing divergent annotations of orthologous transposable elements in related species. We systematically extracted and compared 68,097 Alu insertions in various primates looking for potential events of TE gene conversion and discovered 98 clear cases of Alu–Alu gene conversion, including 64 cases for which the direction of conversion was identified (e.g., AluS conversion to AluY). Gene conversion also does not necessarily affect the entire homologous sequence, and we detected 69 cases of partial gene conversion that resulted in virtual hybrids of two elements. Phylogenetic screening of gene-converted Alus revealed three clear hotspots of the process in the ancestors of Catarrhini, Hominoidea, and gibbons. In general, our systematic screening of orthologous primate loci for gene-converted TEs provides a new strategy and view of a post-integrative process that changes the identities of such elements.

Rad52 Oligomeric N-Terminal Domain Stabilizes Rad51 Nucleoprotein Filaments and Contributes to Their Protection against Srs2

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as a template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52–Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

Development of a SCAR Marker Based on SCoT Polymorphisms for Sugarcane Smut Resistance

Sugarcane smut caused by Sporisorium scitanmineumis the most severe sugarcane disease that causes major economic losses in sugarcane production in China, and disease resistance breeding is an important way of preventing and controlling this disease. In this study, BC3F1lines derived from the cross between YC 73-226 and YCE 06-111 were used to generate sugarcane smut-resistant and -susceptible gene pools using bulked segregant analysis (BSA). Eighty-nine random primers of start codon targeted (SCoT) polymorphisms were screened, whereas only primer SCoT44 could stably amplify the specific fragment (HE-Ss44) in the resistant pool. Then, several primer pairs of sequence characterized amplified regions (SCARs) were designed based on the sequence alignment of HE-Ss44 (920 bp), which was recovered after purification, and only one pair of SCAR primers (Ss44-F2/R2, forward: 5'-GGCGGGCACCGTCGAGTCCACAT-3'; reverse: 5'-CCGTCCGTCGG TCTCGTCCTTACG-3') could stably amplify a 400-bp specific band in resistant gene pool and its individuals. A validation test of SCAR marker Ss44-F2/R2 was performed using 34 sugarcane cultivars with known smut resistance, which revealed a selection accuracy of 82.35% between marker detection and known smut resistance. Moreover, Pearson’s correlation analysis also showed that the SCAR marker Ss44-F2/R2 was significantly correlated (r= 0.583, P= 0.0003 < 0.01) with the smut resistance trait in sugarcane. In addition, the nucleotide sequence of HE-Ss44 linked with smut-resistancewas not aligned to the homologous sequence in GenBank (NCBI), and the accession number was MG740763. The SCAR marker Ss44-F2/R2 developed in this study can be used for the rapid detection of smut resistance in sugarcane and may be utilized as reference for the improvement of sugarcane smut resistance based on molecular marker-assisted selection.© 2021 Friends Science Publishers