Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing.

In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution.

Species-specific variation in mitochondrial genome tandem repeat polymorphisms in hares (Lepus spp., Lagomorpha, Leporidae) provides insight into their evolution.

The non-coding regions of the mitochondrial DNAs (mtDNAs) of hares, rabbits, and pikas (Lagomorpha) contain short (∼20 bp) and long (130-160 bp) tandem repeats, absent in related mammalian orders. In the presented study, we provide in-depth analysis for mountain hare (Lepus timidus) and brown hare (L. europaeus) mtDNA non-coding regions, together with a species- and population-level analysis of tandem repeat variation. Mountain hare short tandem repeats (SRs) as well as other analyzed hare species consist of two conserved 10 bp motifs, with only brown hares exhibiting a single, more variable motif. Long tandem repeats (LRs) also differ in sequence and copy number between species. Mountain hares have four to seven LRs, median value five, while brown hares exhibit five to nine LRs, median value six. Interestingly, introgressed mountain hare mtDNA in brown hares obtained an intermediate LR length distribution, with median copy number being the same as with conspecific brown hare mtDNA. In contrast, transfer of brown hare mtDNA into cultured mtDNA-less mountain hare cells maintained the original LR number, whereas the reciprocal transfer caused copy number instability, suggesting that cellular environment rather than the nuclear genomic background plays a role in the LR maintenance. Due to their dynamic nature and separation from other known conserved sequence elements on the non-coding region of hare mitochondrial genomes, the tandem repeat elements likely to represent signatures of ancient genetic rearrangements. clarifying the nature and dynamics of these rearrangements may shed light on the possible role of NCR repeated elements in mitochondria and in species evolution.

Nucleotide and dinucleotide preference of segmented viruses are shaped more by segment: In case study of tomato spotted wilt virus.

Several studies have showed that the nucleotide and dinucleotide composition of viruses possibly follows their host species or protein coding region. Nevertheless, the influence of viral segment on viral nucleotide and dinucleotide composition is still unknown. Here, we explored through tomato spotted wilt virus (TSWV), a segmented virus that seriously threatens the production of tomatoes all over the world. Through nucleotide composition analysis, we found the same over-representation of A across all viral segments at the first and second codon position, but it exhibited distinct in segments at the third codon position. Interestingly, the protein coding regions which encoded by the same or different segments exhibit obvious distinct nucleotide preference. Then, we found that the dinucleotides UpG and CpU were overrepresented and the dinucleotides UpA, CpG and GpU were underrepresented, not only in the complete genomic sequences, but also in different segments, protein coding regions and host species. Notably, 100% of the data investigated here were predicted to the correct viral segment and protein coding region, despite the fact that only 67% of the data analyzed here were predicted to the correct viral host species. In conclusion, in case study of TSWV, nucleotide composition and dinucleotide preference of segment viruses are more strongly dependent on segment and protein coding region than on host species. This research provides a novel perspective on the molecular evolutionary mechanisms of TSWV and provides reference for future research on genetic diversity of segmented viruses.

Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions.

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.

C and G are frequently mutated into T and A in coding regions of human genes.

Nucleotide mutations in human genes have long been a hot subject for study because some of them may lead to severe human diseases. Understanding the general mutational process and evolutionary trend of human genes could help answer such questions as why certain diseases occur and what challenges we face in protecting human health. In this study, we conducted statistics on 89,895 single-nucleotide variations identified in coding regions of 18,339 human genes. The results show that C and G are frequently mutated into T and A in human genes. C/G (C or G)-to-T/A mutations lead to reduction of hydrogen bonds in double-stranded DNA because C-G and T-A base pairs are maintained by three and two hydrogen bonds respectively. C-to-T and G-to-A mutations occur predominantly in human genes because they not only reduce hydrogen bonds but also belong to transition mutation. Reduction of hydrogen bonds could reduce energy consumption not only in separating double strands of mutated DNA for transcription and replication but also in disrupting stem-loop structure of mutated mRNA for translation. It is thus considered that to reduce hydrogen bonds (and thus to reduce energy consumption in gene expression) is one of the driving forces for nucleotide mutation. Moreover, codon mutation is positively correlated to its content, suggesting that most mutations are not targeted on changing any specific codons (amino acids) but are merely for reducing hydrogen bonds. Our study provides an example of utilizing single-nucleotide variation data to infer evolutionary trend of human genes, which can be referenced to conduct similar studies in other organisms.

cirCodAn: A GHMM-based tool for accurate prediction of coding regions in circRNA.

Studies focusing on characterizing circRNAs with the potential to translate into peptides are quickly advancing. It is helping to elucidate the roles played by circRNAs in several biological processes, especially in the emergence and development of diseases. While various tools are accessible for predicting coding regions within linear sequences, none have demonstrated accurate open reading frame detection in circular sequences, such as circRNAs. Here, we present cirCodAn, a novel tool designed to predict coding regions in circRNAs. We evaluated the performance of cirCodAn using datasets of circRNAs with strong translation evidence and showed that cirCodAn outperformed the other tools available to perform a similar task. Our findings demonstrate the applicability of cirCodAn to identify coding regions in circRNAs, which reveals the potential of use of cirCodAn in future research focusing on elucidating the biological roles of circRNAs and their encoded proteins. cirCodAn is freely available at https://github.com/denilsonfbar/cirCodAn.

Single Nucleotide Polymorphisms [SNPs] in the Shadows: Uncovering their Function in Non-Coding Region of Esophageal Cancer.

Esophageal cancer is a complex disease influenced by genetic and environmental factors. Single nucleotide polymorphisms [SNPs] in non-coding regions of the genome have emerged as crucial contributors to esophageal cancer susceptibility. This review provides a comprehensive overview of the role of SNPs in non-coding regions and their association with esophageal cancer. The accumulation of SNPs in the genome has been implicated in esophageal cancer risk. Various studies have identified specific locations in the genome where SNPs are more likely to occur, suggesting a location-specific response. Chromatin conformational studies have shed light on the localization of SNPs and their impact on gene transcription, posttranscriptional modifications, gene expression regulation, and histone modification. Furthermore, miRNA-related SNPs have been found to play a significant role in esophageal squamous cell carcinoma [ESCC]. These SNPs can affect miRNA binding sites, thereby altering target gene regulation and contributing to ESCC development. Additionally, the risk of ESCC has been linked to base excision repair, suggesting that SNPs in this pathway may influence disease susceptibility. Somatic DNA segment alterations and modified expression quantitative trait loci [eQTL] have also been associated with ESCC. These alterations can lead to disrupted gene expression and cellular processes, ultimately contributing to cancer development and progression. Moreover, SNPs have been found to be associated with the long non-coding RNA HOTAIR, which plays a crucial role in ESCC pathogenesis. This review concludes with a discussion of the current and future perspectives in the field of SNPs in non-coding regions and their relevance to esophageal cancer. Understanding the functional implications of these SNPs may lead to the identification of novel therapeutic targets and the development of personalized approaches for esophageal cancer prevention and treatment.

Genome-wide patterns of non-coding sequence variation in the major fungal pathogen Aspergillus fumigatus.

A.fumigatus is a deadly fungal pathogen, responsible for >400,000 infections/year and high mortality rates. A. fumigatus strains exhibit variation in infection-relevant traits, including in their virulence. However, most A. fumigatus protein-coding genes, including those that modulate its virulence, are shared between A. fumigatus strains and closely related non-pathogenic relatives. We hypothesized that A. fumigatus genes exhibit substantial genetic variation in the non-coding regions immediately upstream to the start codons of genes, which could reflect differences in gene regulation between strains. To begin testing this hypothesis, we identified 5,812 single-copy orthologs across the genomes of 263 A. fumigatus strains. A. fumigatus non-coding regions showed higher levels of sequence variation compared to their corresponding protein-coding regions. Specifically, we found that 1,274 non-coding regions exhibited <75% nucleotide sequence similarity (compared to 928 protein-coding regions) and 3,721 non-coding regions exhibited between 75% and 99% similarity (compared to 2,482 protein-coding regions) across strains. Only 817 non-coding regions exhibited ≥99% sequence similarity compared to 2,402 protein-coding regions. By examining 2,482 genes whose protein-coding sequence identity scores ranged between 75% and 99%, we identified 478 total genes with signatures of positive selection only in their non-coding regions and 65 total genes with signatures only in their protein-coding regions. 28 of the 478 non-coding regions and 5 of the 65 protein-coding regions under selection are associated with genes known to modulate A. fumigatus virulence. Non-coding region variation between A. fumigatus strains included single nucleotide polymorphisms and insertions or deletions of at least a few nucleotides. These results show that non-coding regions of A. fumigatus genes harbor greater sequence variation than protein-coding regions, raising the hypothesis that this variation may contribute to A. fumigatus phenotypic heterogeneity.

An mRNA profiling assay incorporating coding region InDels for body fluid identification and the inference of the donor in mixed samples.

Biological traces discovered at crime scenes hold significant significance in forensic investigations. In cases involving mixed body fluid stains, the evidentiary value of DNA profiles depends on the type of body fluid from which the DNA was obtained. Recently, coding region polymorphism analysis has proved to be a promising method for directly linking specific body fluids to their respective DNA contributors in mixtures, which may help to avoid "association fallacy" between separate DNA and RNA evidence. In this study, we present an update on previously reported coding region Single Nucleotide Polymorphisms (cSNPs) by exploring the potential application of coding region Insertion/Deletion polymorphisms (cInDels). Nine promising cInDels, selected from 70 mRNA markers based on stringent screening criteria, were integrated into an existing mRNA profiling assay. Subsequently, the body fluid specificity of our cInDel assay and the genotyping consistency between complementary DNA (cDNA) and genomic DNA (gDNA) were examined. Our study demonstrates that cInDels can function as important multifunctional genetic markers, as they provide not only the ability to confirm the presence of forensically relevant body fluids, but also the ability to associate/dissociate specific body fluids with particular donors.

Molecular landscape of the JAK2 gene in chronic myeloproliferative neoplasm patients from the state of Amazonas, Brazil.

JAK2V617F (dbSNP: rs77375493) is the most frequent and most-studied variant in BCR::ABL1 negative myeloproliferative neoplasms and in the JAK2 gene. The present study aimed to molecularly characterize variants in the complete coding region of the JAK2 gene in patients with BCR::ABL1 negative chronic myeloproliferative neoplasms. The study included 97 patients with BCR::ABL1 negative myeloproliferative neoplasms, including polycythemia vera (n=38), essential thrombocythemia (n=55), and myelofibrosis (n=04). Molecular evaluation was performed using conventional PCR and Sanger sequencing to detect variants in the complete coding region of the JAK2 gene. The presence of missense variants in the JAK2 gene including rs907414891, rs2230723, rs77375493 (JAK2V617F), and rs41316003 were identified. The coexistence of variants was detected in polycythemia vera and essential thrombocythemia. Thus, individuals with high JAK2V617F variant allele frequency (≥50% VAF) presented more thrombo-hemorrhagic events and manifestations of splenomegaly compared with those with low JAK2V617F variant allele frequency (<50% VAF). In conclusion, individuals with BCR::ABL1 negative neoplasms can display >1 variant in the JAK2 gene, especially rs2230722, rs2230724, and rs77375493 variants, and those with high JAK2V617F VAF show alterations in the clinical-laboratory profile compared with those with low JAK2V617F VAF.

Application of SNaPshot Technology in Semen-Specific cSNP Genetic Marker.

OBJECTIVES: To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains and mixed body fluid identification. METHODS: Genomic DNA (gDNA) and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA (cDNA). The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis (CE). RESULTS: A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the TGM4 gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor. CONCLUSIONS: The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen (stains) and provide information for determining the origin of semen in mixed body fluids (stains).

PDIVAS: Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing.

BACKGROUND: Deep-intronic variants that alter RNA splicing were ineffectively evaluated in the search for the cause of genetic diseases. Determination of such pathogenic variants from a vast number of deep-intronic variants (approximately 1,500,000 variants per individual) represents a technical challenge to researchers. Thus, we developed a Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing (PDIVAS) to easily detect pathogenic deep-intronic variants. RESULTS: PDIVAS was trained on an ensemble machine-learning algorithm to classify pathogenic and benign variants in a curated dataset. The dataset consists of manually curated pathogenic splice-altering variants (SAVs) and commonly observed benign variants within deep introns. Splicing features and a splicing constraint metric were used to maximize the predictive sensitivity and specificity, respectively. PDIVAS showed an average precision of 0.92 and a maximum MCC of 0.88 in classifying these variants, which were the best of the previous predictors. When PDIVAS was applied to genome sequencing analysis on a threshold with 95% sensitivity for reported pathogenic SAVs, an average of 27 pathogenic candidates were extracted per individual. Furthermore, the causative variants in simulated patient genomes were more efficiently prioritized than the previous predictors. CONCLUSION: Incorporating PDIVAS into variant interpretation pipelines will enable efficient detection of disease-causing deep-intronic SAVs and contribute to improving the diagnostic yield. PDIVAS is publicly available at https://github.com/shiro-kur/PDIVAS .

Mitochondrial DNA sequencing of Kehilan and Hamdani horses from Saudi Arabia.

The Arabian horse breed is well known for its purity and played a key role in the genetic improvement of other horses worldwide. The mitochondrial genome plays a vital role in maternal inheritance and it's helpful to evaluate its genetic diversity and conservation. It has higher mutation rates than nuclear DNA in vertebrates and therefore reveals phylogenetic relationships and haplotypes. In this study, the mitochondrial genome mutations in two Saudi horse strains, Kehilan and Hamdani demonstrated various changes in the gene and amino acid levels and included two other Saudi horses (Hadban and Seglawi) from the previous study for phylogenetic comparison. The whole mitochondrial genome sequencing resulted in intra and inter mtDNA variations between the studied horses. Interestingly, the Hamdani horse has nucleotide substitutions similar to those of the Hadban horse, which is reflected in the phylogenetic tree as a significantly close relationship. This type of study provides a better understanding of mitogenome structure and conservation of livestock species genetic data.

DNA:RNA Hybrids Are Major Dinoflagellate Minicircle Molecular Types.

Peridinin-containing dinoflagellate plastomes are predominantly encoded in nuclear genomes, with less than 20 essential chloroplast proteins carried on "minicircles". Each minicircle generally carries one gene and a short non-coding region (NCR) with a median length of approximately 400-1000 bp. We report here differential nuclease sensitivity and two-dimensional southern blot patterns, suggesting that dsDNA minicircles are in fact the minor forms, with substantial DNA:RNA hybrids (DRHs). Additionally, we observed large molecular weight intermediates, cell-lysate-dependent NCR secondary structures, multiple bidirectional predicted ssDNA structures, and different southern blot patterns when probed with different NCR fragments. In silico analysis suggested the existence of substantial secondary structures with inverted repeats (IR) and palindrome structures within the initial ~650 bp of the NCR sequences, in accordance with conversion event(s) outcomes with PCR. Based on these findings, we propose a new transcription-templating-translation model, which is associated with cross-hopping shift intermediates. Since dinoflagellate chloroplasts are cytosolic and lack nuclear envelope breakdown, the dynamic DRH minicircle transport could have contributed to the spatial-temporal dynamics required for photosystem repair. This represents a paradigm shift from the previous understanding of "minicircle DNAs" to a "working plastome", which will have significant implications for its molecular functionality and evolution.

A conventional PCR-based method to detect the E2 gene of the rubella virus for epidemiological analysis.

To eliminate the rubella virus (RV), genetic characterization is vital for its detection, identification of endemic transmission, and diagnosis of imported cases. The 739-nucleotide region in the E1 gene has primarily been used for genotyping for epidemiological analysis. However, in the 2018-2019 RV outbreak, identical sequences were observed in patients who were not epidemiologically linked. Additionally, the 739 nt sequences from the outbreak in Tokyo in 2018-2019 were identical to RV identified in China in 2019. This suggests that this region may be insufficient to identify the detected RV strains as endemic or imported. In 62.4% of the specimens, the E1 gene sequences of the 1E RV genotype were identical. Additionally, the observed discordance of sequences from the mainly detected identical sequence in the 739-nt sequence of the E1 gene were one (31.0%), two (3.5%), three (2.6%), and four (0.23%). Moreover, a comparison of the complete structural protein-coding region suggests that the E2 gene is more diverse than the E1 and the capsid gene. Thus, conventional polymerase chain reaction (PCR) primers were developed to detect the E2 gene and improve epidemiological analysis. A comparison of the sequences identified during the RV outbreak in Tokyo revealed genetic differences in the sequences (15 of the 18 specimens). These results suggest that additional information could be obtained by simultaneously analyzing the E2 and the E1 region. The identified sequences can potentially aid in evaluating the RV strains detected during epidemiological analysis.

Investigation of UTR Variants by Computational Approaches Reveal Their Functional Significance in PRKCI Gene Regulation.

Single nucleotide polymorphisms (SNPs) are associated with many diseases including neurological disorders, heart diseases, diabetes, and different types of cancers. In the context of cancer, the variations within non-coding regions, including UTRs, have gained utmost importance. In gene expression, translational regulation is as important as transcriptional regulation for the normal functioning of cells; modification in normal functions can be associated with the pathophysiology of many diseases. UTR-localized SNPs in the PRKCI gene were evaluated using the PolymiRTS, miRNASNP, and MicroSNIper for association with miRNAs. Furthermore, the SNPs were subjected to analysis using GTEx, RNAfold, and PROMO. The genetic intolerance to functional variation was checked through GeneCards. Out of 713 SNPs, a total of thirty-one UTR SNPs (three in 3' UTR region and twenty-nine in 5' UTR region) were marked as ≤2b by RegulomeDB. The associations of 23 SNPs with miRNAs were found. Two SNPs, rs140672226 and rs2650220, were significantly linked with expression in the stomach and esophagus mucosa. The 3' UTR SNPs rs1447651774 and rs115170199 and the 5' UTR region variants rs778557075, rs968409340, and 750297755 were predicted to destabilize the mRNA structure with substantial change in free energy (∆G). Seventeen variants were predicted to have linkage disequilibrium with various diseases. The SNP rs542458816 in 5' UTR was predicted to put maximum influence on transcription factor binding sites. Gene damage index(GDI) and loss of function (o:e) ratio values for PRKCI suggested that the gene is not tolerant to loss of function variants. Our results highlight the effects of 3' and 5' UTR SNP on miRNA, transcription and translation of PRKCI. These analyses suggest that these SNPs can have substantial functional importance in the PRKCI gene. Future experimental validation could provide further basis for the diagnosis and therapeutics of various diseases.

Clinical profile and molecular typing of viral etiological agents associated with Hand, Foot and Mouth Disease (HFMD): A study from Udhampur, Northern India.

PURPOSE: Hand, Foot and Mouth disease (HFMD) is a contagious pediatric viral disease caused due to enteroviruses (EV) of the family Picornaviridae. Cases of HFMD were reported from a tertiary care health centre, Udhampur, (Jammu and Kashmir), Northern India. The present study highlights the clinical and molecular virological aspects of HFMD cases. MATERIAL AND METHODS: Cases reported during August 2016-September 2017, and clinically diagnosed as HFMD of all age groups were included. Clinical, Biochemical and molecular virology aspects were compared. Clinical samples (n â€‹= â€‹50) such as vesicle swab, buccal and throat swabs were collected for enterovirus detection. EV-RNA was detected by 5'NCR based RT-PCR and genotyping by VP1 gene amplification and cycle sequencing. RESULTS: Of the cases of HFMD enrolled (n â€‹= â€‹50), highest (84%) were of children aged <5 years, presented either or both anathemas and exanthemas with prodromal symptoms (fever, irritability). Clinical presentations involved mainly oral ulcers on lips and tongue (48%). Oral erosions were either single or multiple in numbers. Exanthemas were seen on hand and palm, widely spread up to buttocks, legs, arms and trunk. Of these, six patients were found anemic. Complete blood count (CBC) indicated lymphocytosis and C-reactive protein (n â€‹= â€‹10) in children aged <5 years. EV-RNA was detected in 78% (39/50) of the clinical samples. VP1 gene based typing indicated the presence of CV-A16, CVA6 and EV-A71 types. CONCLUSIONS: The study highlights association of EVs in HFMD cases in the reported region. CV-A16, CV-A6 and EV-A71 types were reported for the first time from Udhampur (J&K), Northern India. No differences were observed in the clinical profile of EV strains detected. Circulation of the strains warrant and alarm outbreaks. More focused studies on HFMD and monitoring of viral strains is mandatory.

Application of SNaPshot Technology in Semen-Specific cSNP Genetic Marker / 法医学杂志

OBJECTIVES@#To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains and mixed body fluid identification.@*METHODS@#Genomic DNA (gDNA) and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA (cDNA). The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis (CE).@*RESULTS@#A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the TGM4 gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor.@*CONCLUSIONS@#The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen (stains) and provide information for determining the origin of semen in mixed body fluids (stains).

Bacterial Transcription Factors Bind to Coding Regions and Regulate Internal Cryptic Promoters.

Transcription factors (TFs) regulate transcription by binding to the specific sequences at the promoter region. However, the mechanisms and functions of TFs binding within the coding sequences (CDS) remain largely elusive in prokaryotes. To this end, we collected 409 data sets for bacterial TFs, including 104 chromatin immunoprecipitation sequencing (ChIP-seq) assays and 305 data sets from the systematic evolution of ligands by exponential enrichment (SELEX) in seven model bacteria. Interestingly, these TFs displayed the same binding capabilities for both coding and intergenic regions. Subsequent biochemical and genetic experiments demonstrated that several TFs bound to the coding regions and regulated the transcription of the binding or adjacent genes. Strand-specific RNA sequencing revealed that these CDS-binding TFs regulated the activity of the cryptic promoters, resulting in the altered transcription of the corresponding antisense RNA. TF RhpR hindered the transcriptional elongation of a subgenic transcript within a CDS. A ChIP-seq and Ribo-seq coanalysis revealed that RhpR influenced the translational efficiency of binding genes. Taken together, the present study reveals three regulatory mechanisms of CDS-bound TFs within individual genes, operons, and antisense RNAs, which demonstrate the variability of the regulatory mechanisms of TFs and expand upon the complexity of bacterial transcriptomes. IMPORTANCE Although bacterial TFs regulate transcription by binding to specific sequences at the promoter region, little is known about the mechanisms and functions of TFs binding within the CDS. In this study, we show that bacterial TFs have same binding pattern in both CDS and promoter regions, and we reveal three regulatory mechanisms of CDS-bound TF that together demonstrate the complexity of the regulatory mechanisms of bacterial TFs and the wide spread of internal cryptic promoters in CDS.

Structural Characteristics of the 5'-Terminal Region of Mouse p53 mRNA and Identification of Proteins That Bind to This mRNA Region.

A mouse model has often been used in studies of p53 gene expression. Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA's structure and its interactions with proteins in the translation process. In particular, the 5'-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5'-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5'-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5'-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.