Development of DNA Insertion-specific Markers Based on the Intergenic Region of Oplegnathus punctatus Cdkn1/srsf3 for Sex Identification.

Spotted knifejaw (Oplegnathus punctatus) is a marine economic fish with high food and ecological value, and its growth process has obvious male and female sexual dimorphism, with males growing significantly faster than females. However, the current sex identification technology is not yet mature, which will limit the growth rate of O. punctatus aquaculture and the efficiency of separate sex breeding, so the development of efficient sex molecular markers is imperative. This study identified a 926 bp DNA insertion fragment in the cdkn1/srsf3 intergenic region of O. punctatus males through whole-genome scanning, comparative genomics, and structural variant analysis. A pair of primers was designed based on the insertion information of the Y chromosome intergenic region in male individuals. Agarose gel electrophoresis revealed the amplification of two DNA fragments, 1118 bp and 192 bp, in male O. punctatus individuals. The 926 bp fragment was identified as the insertion in the intergenic region of cdkn1/srsf3 in males, while only a single 192 bp DNA fragment was amplified in females. The biological sex of the individuals identified in this manner was consistent with their known phenotypic sex. In this study, we developed a method to detect DNA insertion variants in the intergenic region of O. punctatus. Additionally, we introduced a new DNA marker for the rapid identification of the sex of O. punctatus, which enhances detection efficiency. The text has important reference significance and application value in sex identification, all-male breeding, and lineage selection. It provides new insights into the regulation of variation in the intergenic region of cdkn1/srsf3 genes and the study of RNA shearing.

The first ITS1 profiling of honey samples from the Southeast Asian region Lombok, Bali and Banggi Island.

Southern Asian flowers offer honeybees a diversity of nectar. Based on its geographical origin, honey quality varies. Traditional methods are less authentic than DNA-based identification. The origin of honey is determined by pollen, polyphenolic, and macro-microorganisms. In this study, amplicon sequencing targets macro-microorganisms in eDNA using the ITS1 region to explore honey's geographical location and authentication. The variety of honey samples was investigated using ITS1 with Illumina sequencing. For all four honey samples, raw sequence reads showed 979,380 raw ITS1 amplicon reads and 375 ASVs up to the phylum level. The highest total number of 202 ASVs up to phylum level identified Bali honey with 211,189 reads, followed by Banggi honey with 309,207 a total number of 111 ASVs, and Lombok represents only 63 ASVs up to phylum level with several read 458,984. Based on Shannon and Chao1, honey samples from Bali (B2) and (B3) exhibited higher diversity than honey from Lombok (B1) and green honey from Sabah (B4), while the Simpson index showed that Banggi honey (B4) had higher diversity. Honey samples had significant variance in mycobiome taxonomic composition and abundance. Zygosaccharomyces and Aspergillus were the main genera found in Lombok honey, with percentages of 68.81% and 29.76% respectively. Bali honey samples (B2 and B3) were identified as having a significant amount of the genus Aureobasidium, accounting for 40.81% and 25% of the readings, respectively. The microbiome composition of Banggi honey (B4) showed a high presence of Zygosaccharomyces 45.17% and Aureobasidium 35.24%. The ITS1 analysis effectively distinguishes between honey samples of different origins and its potential as a discriminatory tool for honey origin and authentication purposes.

Engineering spacer specificity of the Cre/loxP system.

Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing Cre discrimination between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the plasticity of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations were used to investigate the possible mechanisms behind the specificity switch. Our findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.

Intergenic sequences harboring potential enhancer elements contribute to Axenfeld-Rieger syndrome by regulating PITX2.

Recent studies have uncovered that noncoding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese family with ARS and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between paired-like homeodomain transcription factor 2 (PITX2) and family with sequence similarity 241 member A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-Seq and real-time quantitative PCR revealed a significant reduction in Pitx2 gene expression in LOH-1-/- mice, while forkhead box C1 expression remained unchanged. ChIP-Seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a substantial downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) that is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence that is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent.

The discovery of novel noncoding RNAs in 50 bacterial genomes.

Structured noncoding RNAs (ncRNAs) contribute to many important cellular processes involving chemical catalysis, molecular recognition and gene regulation. Few ncRNA classes are broadly distributed among organisms from all three domains of life, but the list of rarer classes that exhibit surprisingly diverse functions is growing. We previously developed a computational pipeline that enables the near-comprehensive identification of structured ncRNAs expressed from individual bacterial genomes. The regions between protein coding genes are first sorted based on length and the fraction of guanosine and cytidine nucleotides. Long, GC-rich intergenic regions are then examined for sequence and structural similarity to other bacterial genomes. Herein, we describe the implementation of this pipeline on 50 bacterial genomes from varied phyla. More than 4700 candidate intergenic regions with the desired characteristics were identified, which yielded 44 novel riboswitch candidates and numerous other putative ncRNA motifs. Although experimental validation studies have yet to be conducted, this rate of riboswitch candidate discovery is consistent with predictions that many hundreds of novel riboswitch classes remain to be discovered among the bacterial species whose genomes have already been sequenced. Thus, many thousands of additional novel ncRNA classes likely remain to be discovered in the bacterial domain of life.

Genome composition and GC content influence loci distribution in reduced representation genomic studies.

BACKGROUND: Genomic architecture is a key evolutionary trait for living organisms. Due to multiple complex adaptive and neutral forces which impose evolutionary pressures on genomes, there is a huge variability of genomic features. However, their variability and the extent to which genomic content determines the distribution of recovered loci in reduced representation sequencing studies is largely unexplored. RESULTS: Here, by using 80 genome assemblies, we observed that whereas plants primarily increase their genome size by expanding their intergenic regions, animals expand both intergenic and intronic regions, although the expansion patterns differ between deuterostomes and protostomes. Loci mapping in introns, exons, and intergenic categories obtained by in silico digestion using 2b-enzymes are positively correlated with the percentage of these regions in the corresponding genomes, suggesting that loci distribution mostly mirrors genomic architecture of the selected taxon. However, exonic regions showed a significant enrichment of loci in all groups regardless of the used enzyme. Moreover, when using selective adaptors to obtain a secondarily reduced loci dataset, the percentage and distribution of retained loci also varied. Adaptors with G/C terminals recovered a lower percentage of selected loci, with a further enrichment of exonic regions, while adaptors with A/T terminals retained a higher percentage of loci and slightly selected more intronic regions than expected. CONCLUSIONS: Our results highlight how genome composition, genome GC content, RAD enzyme choice and use of base-selective adaptors influence reduced genome representation techniques. This is important to acknowledge in population and conservation genomic studies, as it determines the abundance and distribution of loci.

The internal transcribed spacer 1 sequence polymorphism brings updates to tsetse species distribution in the northern Cameroon: Importance in planning efficient vector control.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning.

Whole genome sequencing identifies associations for nonsyndromic sagittal craniosynostosis with the intergenic region of BMP2 and noncoding RNA gene LINC01428.

Craniosynostosis (CS) is a major birth defect resulting from premature fusion of cranial sutures. Nonsyndromic CS occurs more frequently than syndromic CS, with sagittal nonsyndromic craniosynostosis (sNCS) presenting as the most common CS phenotype. Previous genome-wide association and targeted sequencing analyses of sNCS have identified multiple associated loci, with the strongest association on chromosome 20. Herein, we report the first whole-genome sequencing study of sNCS using 63 proband-parent trios. Sequencing data for these trios were analyzed using the transmission disequilibrium test (TDT) and rare variant TDT (rvTDT) to identify high-risk rare gene variants. Sequencing data were also examined for copy number variants (CNVs) and de novo variants. TDT analysis identified a highly significant locus at 20p12.3, localized to the intergenic region between BMP2 and the noncoding RNA gene LINC01428. Three variants (rs6054763, rs6054764, rs932517) were identified as potential causal variants due to their probability of being transcription factor binding sites, deleterious combined annotation dependent depletion scores, and high minor allele enrichment in probands. Morphometric analysis of cranial vault shape in an unaffected cohort validated the effect of these three single nucleotide variants (SNVs) on dolichocephaly. No genome-wide significant rare variants, de novo loci, or CNVs were identified. Future efforts to identify risk variants for sNCS should include sequencing of larger and more diverse population samples and increased omics analyses, such as RNA-seq and ATAC-seq.

The Density of Regulatory Information Is a Major Determinant of Evolutionary Constraint on Noncoding DNA in Drosophila.

Evolutionary analyses have estimated that ∼60% of nucleotides in intergenic regions of the Drosophila melanogaster genome are functionally relevant, suggesting that regulatory information may be encoded more densely in intergenic regions than has been revealed by most functional dissections of regulatory DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the ∼90 kb of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory information that drives svb expression in the abdominal pupal epidermis is organized in a different way than the elements that drive svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact enhancers separated by large inactive DNA regions, svb expression in the pupal epidermis is driven by regulatory information distributed over broader regions of svb cis-regulatory DNA. In the same vein, we observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved noncoding DNA of the Drosophila genome is contained within regions of open chromatin. These results suggest that part of the evolutionary constraint on noncoding DNA of Drosophila is explained by the density of regulatory information, which may be greater than previously appreciated.

Mitochondrial genome of Hancornia speciosa gomes: intergenic regions containing retrotransposons and predicted genes.

BACKGROUND: Plant mitochondrial genomes are characterized by high homologous recombination, extensive intergenic spacers, conservation in DNA sequences, and gene content. The Hancornia genus belongs to the Apocynaceae family, with H. speciosa Gomes being the sole species in the genus. It is an siganificant commercial fruit crop; however, only a number of studies have been conducted. In this study, we present the mitochondrial genome of H. speciosa and compare it with other mitochondrial genomes within the Apocynaceae family. METHODS AND RESULTS: A total of 2.8 Gb of Illumina paired-end reads were used to obtain the mitogenome, resulting in 22 contigs that were merged using 6.1 Gb of Illumina mate-pair reads to obtain a circular chromosome. The mitochondrial genome of H. speciosa is circular, containing 63 predicted functional genes, spanning a length of 741,811 bp, with a CG content of 44%. Within the mitogenome, 50 chloroplast DNA sequences, equivalent to 1.72% of the genome, were detected. However, intergenic spaces accounted for 703,139 bp (94.79% of the genome), and 287 genes were predicted, totaling 173,721 bp. CONCLUSION: This suggests the incorporation of nuclear DNA into the mitogenome of H. speciosa and self duplication. Comparative analysis among the mitogenomes in the Apocynaceae family revealed a diversity in the structure mediated by recombination, with similar gene content and large intergenic spaces.

Genetic Modification of a Hox Locus Drives Mimetic Color Pattern Variation in a Highly Polymorphic Bumble Bee.

Müllerian mimicry provides natural replicates ideal for exploring mechanisms underlying adaptive phenotypic divergence and convergence, yet the genetic mechanisms underlying mimetic variation remain largely unknown. The current study investigates the genetic basis of mimetic color pattern variation in a highly polymorphic bumble bee, Bombus breviceps (Hymenoptera, Apidae). In South Asia, this species and multiple comimetic species converge onto local Müllerian mimicry patterns by shifting the abdominal setal color from orange to black. Genetic crossing between the orange and black phenotypes suggested the color dimorphism being controlled by a single Mendelian locus, with the orange allele being dominant over black. Genome-wide association suggests that a locus at the intergenic region between 2 abdominal fate-determining Hox genes, abd-A and Abd-B, is associated with the color change. This locus is therefore in the same intergenic region but not the same exact locus as found to drive red black midabdominal variation in a distantly related bumble bee species, Bombus melanopygus. Gene expression analysis and RNA interferences suggest that differential expression of an intergenic long noncoding RNA between abd-A and Abd-B at the onset setal color differentiation may drive the orange black color variation by causing a homeotic shift late in development. Analysis of this same color locus in comimetic species reveals no sequence association with the same color shift, suggesting that mimetic convergence is achieved through distinct genetic routes. Our study establishes Hox regions as genomic hotspots for color pattern evolution in bumble bees and demonstrates how pleiotropic developmental loci can drive adaptive radiations in nature.

Intergenic spacer (IGS-1) region sequence-based identification, genotypic analysis, and antifungal susceptibility of clinical Trichosporon species.

OBJECTIVES: Molecular genotyping of Trichosporon species using intergenic spacer region (IGS-1) sequencing and antifungal drug susceptibility testing of T. asahii clinical isolates from Indian patients. MATERIALS AND METHODS: Fifty-five Trichosporon strains were characterized using IGS-1 sequencing from 2006 to 2018 and tested against 5 antifungals using CLSI M27-A3 guidelines. RESULTS: In this study, broad-spectrum antibiotics with steroids, catheters, and ICU stays were major underlying risk factors. These cases were most commonly associated with diabetes (type-2), chronic obstructive pulmonary disease, and hypertension. Out of fifty-five isolates, 47 (85%) were identified as T. asahii, and the remaining 6 were T. inkin (11%) and 2 were Cutaneotrichosporon dermatis (3.6%). The most common genotype of T. asahii was G3 (22; 49%) subsequently G4 (12; 23%), G1 (8; 17%), and G7 (2; 4%). One new genotype of T asahii was found in addition to the fifteen already known genotypes. Indian T. asahii isolates showed a low level of amphotericin B (range 0.06-4 â€‹mg/l) resistance but relatively higher in fluconazole (range 0.25-64 â€‹mg/l). Although, comparatively low MIC ranges were found in the case of voriconazole (0.03-1 â€‹mg/l), posaconazole (0.06-1 â€‹mg/l) and itraconazole (0.06-1 â€‹mg/l). Voriconazole appeared to be the most active drug in T. asahii isolates. The MICs for all the drugs were comparatively lower in the case of non-Trichosporon asahii strains. CONCLUSION: T. asahii was the most common Trichosporon isolate. Speciation is necessary for optimal antifungal therapy. Voriconazole-based treatment, Steroids, removal of catheters and control of underlying conditions results in positive outcomes.

The structure and mechanism of action of a distinct class of dicistrovirus intergenic region IRESs.

Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of ∼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3'-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem-loop base-paired to mRNA. However, they are ∼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem-loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.

Initiation of translation on nedicistrovirus and related intergenic region IRESs by their factor-independent binding to the P site of 80S ribosomes.

Initiation of translation on many viral mRNAs occurs by noncanonical mechanisms that involve 5' end-independent binding of ribosomes to an internal ribosome entry site (IRES). The ∼190-nt-long intergenic region (IGR) IRES of dicistroviruses such as cricket paralysis virus (CrPV) initiates translation without Met-tRNAi Met or initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the ∼165-nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stem-loop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) that contains a UACUA loop motif and a protruding CrPV-like stem--loop SLIV. In vitro reconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNAi Met Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.

Chance promoter activities illuminate the origins of eukaryotic intergenic transcriptions.

It is debated whether the pervasive intergenic transcription from eukaryotic genomes has functional significance or simply reflects the promiscuity of RNA polymerases. We approach this question by comparing chance promoter activities with the expression levels of intergenic regions in the model eukaryote Saccharomyces cerevisiae. We build a library of over 105 strains, each carrying a 120-nucleotide, chromosomally integrated, completely random sequence driving the potential transcription of a barcode. Quantifying the RNA concentration of each barcode in two environments reveals that 41-63% of random sequences have significant, albeit usually low, promoter activities. Therefore, even in eukaryotes, where the presence of chromatin is thought to repress transcription, chance transcription is prevalent. We find that only 1-5% of yeast intergenic transcriptions are unattributable to chance promoter activities or neighboring gene expressions, and these transcriptions exhibit higher-than-expected environment-specificity. These findings suggest that only a minute fraction of intergenic transcription is functional in yeast.

Rearrangement distance with reversals, indels, and moves in intergenic regions on signed and unsigned permutations.

Genome rearrangement events are widely used to estimate a minimum-size sequence of mutations capable of transforming a genome into another. The length of this sequence is called distance, and determining it is the main goal in genome rearrangement distance problems. Problems in the genome rearrangement field differ regarding the set of rearrangement events allowed and the genome representation. In this work, we consider the scenario where the genomes share the same set of genes, gene orientation is known or unknown, and intergenic regions (structures between a pair of genes and at the extremities of the genome) are taken into account. We use two models, the first model allows only conservative events (reversals and moves), and the second model includes non-conservative events (insertions and deletions) in the intergenic regions. We show that both models result in NP-hard problems no matter if gene orientation is known or unknown. When the information regarding the orientation of genes is available, we present for both models an approximation algorithm with a factor of 2. For the scenario where this information is unavailable, we propose a 4-approximation algorithm for both models.

Hematopoietic/erythroid enhancers activate nearby target genes by extending histone H3K27ac and transcribing intergenic RNA.

Enhancers activate gene transcription remotely, which requires tissue specific transcription factors binding to them. GATA1 and TAL1 are hematopoietic/erythroid-specific factors and often bind together to enhancers, activating target genes. Interestingly, we found that some hematopoietic/erythroid genes are transcribed in a GATA1-dependent but TAL1-independnet manner. They appear to have enhancers within a relatively short distance. In this study, we paired highly transcribed hematopoietic/erythroid genes with the nearest GATA1/TAL1-binding enhancers and analyzed these putative enhancer-gene pairs depending on distance between them. Enhancers located at various distances from genes in the pairs, which was not related to transcription level of the genes. However, genes with enhancers at short distances away tended to be transcriptionally unaffected by TAL1 depletion. Histone H3K27ac extended from the enhancers to target genes. The H3K27ac extension was maintained without TAL1, even though it disappeared owing to the loss of GATA1. Intergenic RNA was highly transcribed from the enhancers to nearby target genes, independent of TAL1. Taken together, TAL1-independent transcription of hematopoietic/erythroid genes appears to be promoted by enhancers present in a short distance. These enhancers are likely to activate nearby target genes by tracking the intervening regions.

Intergenic Regions of Saccharomycotina Yeasts are Enriched in Potential to Encode Transmembrane Domains.

Intergenic genomic regions have essential regulatory and structural roles that impose constraints on their sequences. But regions that do not currently encode proteins also carry the potential to do so in the future. De novo gene emergence, the evolution of novel genes out of previously noncoding sequences has now been established as a potent force for genomic novelty. Recently, it was shown that intergenic regions in the genome of Saccharomyces cerevisiae harbor pervasive cryptic potential to, if theoretically translated, form transmembrane domains (TM domains) more frequently than expected by chance given their nucleotide composition, a property that we refer to as TM-forming enrichment. The source and biological relevance of this property is unknown. Here, we expand the investigation into the TM-forming potential of intergenic regions to the entire Saccharomycotina budding yeast subphylum, in an effort to explain this property and understand its importance. We find pervasive but variable enrichment in TM-forming potential across the subphylum regardless of the composition and average size of intergenic regions. This cryptic property is evenly spread across the genome, cannot be explained by the hydrophobic content of the sequence, and does not appear to localize to regions containing regulatory motifs. This TM-forming enrichment specifically, and not the actual TM-forming potential, is associated, across genomes, with more TM domains in evolutionarily young genes. Our findings shed light on this newly discovered feature of yeast genomes and constitute a first step toward understanding its evolutionary importance.

Transcribed intergenic regions exhibit a lower frequency of nucleotide polymorphism than the untranscribed intergenic regions in the genomes of Escherichia coli and Salmonella enterica.

The temporary exposure of single-stranded regions in the genome during the process of replication and transcription makes the region vulnerable to cytosine deamination resulting in a higher rate of C→T transition. Intraoperon intergenic regions undergo transcription along with adjacent co-transcribed genes in an operon, whereas interoperon intergenic regions are usually devoid of transcription. Hence these two types of intergenic regions (IGRs) can be compared to find out the contribution of replication-associated mutations (RAM) and transcription-associated mutations (TrAM) towards bringing variation in genomes. In our work, we performed a polymorphism spectra comparison between intraoperon IGRs and interoperon IGRs in genomes of two well-known closely related bacteria such as Escherichia coli and Salmonella enterica. In general, the size of intraoperon IGRs was smaller than that of interoperon IGRs in E. coli and S. enterica. Interestingly, the polymorphism frequency at intraoperon IGRs was 2.5-fold lesser than that in the interoperon IGRs in E. coli genome. Similarly, the polymorphism frequency at intraoperon IGRs was 2.8-fold lesser than that in the inter-operon IGRs in S. enterica genome. Therefore, the intraoperon IGRs were often observed to be more conserved. In the case of interoperon IGRs, the T→C transition frequency was a minimum of two times more frequent than T→A transversion frequency whereas in the case of intraoperon IGRs, T→C transition frequency was similar to that of T→A transversion frequency. The polymorphism was purine-biased and keto-biased more in intraoperon IGRs than the inter-operon IGRs. In E. coli, the transition/transversion ratio was observed as 1.639 and 1.338 in inter-operon and in intraoperon IGRs, respectively. In S. enterica, the transition/transversion ratio was observed as 2.134 and 2.780 in inter-operon and in intraoperon IGRs, respectively. The observation in this study indicates that transcribable IGRs might not always have higher polymorphism frequency than nontranscribable IGRs. The lower polymorphism frequency at intraoperon IGRs might be attributed to different events such as the transcription-coupled DNA repair, sequences facilitating translation initiation and avoidance of Rho-dependent transcription termination.