Nucleotide Repeats Research Articles

Identification, distribution and comparative analysis of microsatellites in the chloroplast genome of Oryza species

Chloroplast genome is important because of its maternally inherited, conserved within species and stable in structure. This allows elucidation of inter-specific comparison, identification and establishment of evolutionary relationship among different species. Different classes of DNA elements were present in chloroplast genome of Oryza species, among them chloroplast microsatellites were supposed to be highly variable. In this study, a total of 102 sequences of chloroplast genome of 23 species of Oryza genus belonging to ten different genome types were downloaded from NCBI nucleotide database. The chloroplast DNA length varied from 134,401bp to 136,133bp. Microsatellite analysis revealed that total SSR number varied from 268 in O. coarctata to 281 in O. granulata. Comparative analysis of repeat type revealed that mononucleotide and trinucleotide were least present, while penta and hexa nucleotide motif were the most common type among all the Oryza species. Frequency analysis of the repeat revealed that T repeat among mono, AT among di, AAT among tri, AATA among tetra, AATTC among penta and AAAGAA among hexa nucleotide repeat was found to be common among most of the Oryza species. Total of five set of SSR markers flanking to penta and hexa nucleotide repeat types were developed which uniquely present among species, namely, O. barthi, O. sativa Japonica, O. cocarctata, O. rhizomatis and O. ridley. Phylogeny based on SSR markers indicate separate evolution of different SSR markers among individual species.

Genome survey and characteristic analysis of SSR in Callicarpa nudiflora

Callicarpa nudiflora,which is a big brand of Li nationality medicine with Hainan characteristics,has the effects of dissolving stasis,hemostasis,anti-inflammatory and antibacterial. At present,there is a lack of information about the reference genome of C. nudiflora. The study of the genome size,heterozygosity rate and characteristics of SSR of C. nudiflora,can provide an effective basis for the formulation of the whole genome de novo sequencing strategy and development of SSR molecular markers of C. nudiflora. To realize this purpose,high throughput sequencing platform Illumina Hiseq was used to sequence the genome structure of C. nudiflora and K-mer analysis was applied to estimate genome size,repeat sequences and heterozygosity rate. Simple-sequence repeat( SSR) loci that are suitable as markers were identified by MISA software. The results showed the estimated genome size of C. nudiflora was 822. 43 Mb,with a 0. 85% heterozygosity rate and 71. 67% repeats,and the GC content of genome was about 49. 20%. Therefore,C. nudiflora belongs to a complex genome with high heterozygosity and repetition. SSR molecular genetic markers were analyzed in the genome sequence,and a total of 206 049 SSRs were identified,among which mono-nucleotide,di-nucleotide and tri-nucleotide repetitive motifs summed up to 198 993,accounting for 96. 57% of the total SSRs. Among the 2-6 nucleotide repeats,AT/AT,AAT/ATT,AGCC/CTGG,AAAAT/ATTTT and AGATAT/ATATCT have the largest number,respectively. This report represents the first genome-wide characterization of C. nudiflora,and provides a reference for the construction of the library for the fine sequencing of the genome,and a molecular basis for the development of SSR molecular markers as well as for the protection and utilization of gene resources.

Quantitative Studies of Muscleblind Proteins and Their Interaction With TCF4 RNA Foci Support Involvement in the Mechanism of Fuchs' Dystrophy.

PurposeFuchs' endothelial corneal dystrophy (FECD) is a major cause of vision loss and the most common nucleotide repeat disorder, affecting 4% of United States population greater than 40 years of age. Seventy percent of FECD cases are due to an intronic CTG expansion within the TCF4 gene, resulting in accumulation of CUG repeat RNA nuclear foci in corneal endothelium. Each endothelial cell has approximately two sense foci, and each focus is a single RNA molecule. This study aimed to obtain a better understanding of how rare repeat RNA species lead to disease.MethodsWe quantitatively examined muscleblind-like (MBNL) proteins and their interaction with foci in both patient-derived corneal endothelial cell lines and human corneal endothelial tissue.ResultsUsing fluorescent in situ hybridization and immunofluorescence, we found that depletion of both MBNL1 and MBNL2 reduces nuclear RNA foci formed by the repeat, suggesting that both are necessary for foci. Quantitative studies of RNA and protein copy number revealed MBNLs to be abundant in the total cellular pool in endothelial cell lines but are much lower in human corneal endothelial tissue. Studies using human tissue nuclear and cytoplasmic fractions indicate that most MBNL proteins are localized to the cytoplasm.ConclusionsThe low levels of MBNL1/2 in corneal tissue, in combination with the small fraction of protein in the nucleus, may make corneal endothelial cells especially susceptible to sequestration of MBNL1/2 by CUG repeat RNA. These observations may explain how a limited number of RNA molecules can cause widespread alteration of splicing and late-onset degenerative FECD.

Transcriptome sequencing, de novo assembly, characterisation of wild accession of blackgram (Vigna mungo var. silvestris) as a rich resource for development of molecular markers and validation of SNPs by high resolution melting (HRM) analysis

BackgroundBlackgram [Vigna mungo (L.) Hepper], is an important legume crop of Asia with limited genomic resources. We report a comprehensive set of genic simple sequence repeat (SSR) and single nucleotide polymorphism (SNPs) markers using Illumina MiSeq sequencing of transcriptome and its application in genetic variation analysis and mapping.ResultsTranscriptome sequencing of immature seeds of wild blackgram, V. mungo var. silvestris by Illumina MiSeq technology generated 1.9 × 107 reads, which were assembled into 40,178 transcripts (TCS) with an average length of 446 bp covering 2.97 GB of the genome. A total of 38,753 CDS (Coding sequences) were predicted from 40,178 TCS and 28,984 CDS were annotated through BLASTX and mapped to GO and KEGG database resulting in 140 unique pathways. The tri-nucleotides were most abundant (39.9%) followed by di-nucleotide (30.2%). About 60.3 and 37.6% of SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Among SNPs, the most abundant substitution type were transitions (Ts) (61%) followed by transversions (Tv) type (39%), with a Ts/Tv ratio of 1.58. A total of 2306 DEGs were identified by RNA Seq between wild and cultivar and validation was done by quantitative reverse transcription polymerase chain reaction. In this study, we genotyped SNPs with a validation rate of 78.87% by High Resolution Melting (HRM) Assay.ConclusionIn the present study, 1621genic-SSR and 1844 SNP markers were developed from immature seed transcriptome sequence of blackgram and 31 genic-SSR markers were used to study genetic variations among different blackgram accessions. Above developed markers contribute towards enriching available genomic resources for blackgram and aid in breeding programmes.

A New View of the T-Loop Junction: Implications for Self-Primed Telomere Extension, Expansion of Disease-Related Nucleotide Repeat Blocks, and Telomere Evolution.

Telomere loops (t-loops) are formed at the ends of chromosomes in species ranging from humans to worms, plants, and with genetic manipulation, some yeast. Recent in vitro studies demonstrated that transcription of telomeric DNA leads to highly efficient t-loop formation. It was also shown that both DNA termini are inserted into the preceding DNA to generate a highly stable t-loop junction. Furthermore, some telomeric RNA remains present at the junction, potentially acting as a plug to further protect and stabilize the t-loop. Modeling the loop junction reveals two mechanisms by which the canonical chromosomal replication factors could extend the telomere in the absence of telomerase. One mechanism would utilize the annealed 3’ terminus as a de novo replication origin. In vitro evidence for the ability of the t-loop to prime telomere extension using the T7 replication factors is presented. A second mechanism would involve resolution of the Holliday junction present in the t-loop bubble by factors such as GEN1 to generate a rolling circle template at the extreme terminus of the telomere. This could lead to large expansions of the telomeric tract. Here, we propose that telomeres evolved as terminal elements containing long arrays of short nucleotide repeats due to the ability of such arrays to fold back into loops and self-prime their replicative extension. In this view, telomerase may have evolved later to provide a more precise mechanism of telomere maintenance. Both pathways have direct relevance to the alternative lengthening of telomeres (ALT) pathway. This view also provides a possible mechanism for the very large repeat expansions observed in nucleotide repeat diseases such as Fragile X syndrome, myotonic dystrophy, familial amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). The evolution of telomeres is discussed in the framework of these models.

RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats.

Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation (RAN translation) of C9orf72 repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for c9ALS/FTD and other neurodegenerative diseases caused by nucleotide repeat expansions.

Genome-Wide Identification of Microsatellites and Transposable Elements in the Dromedary Camel Genome Using Whole-Genome Sequencing Data.

Transposable elements (TEs) along with simple sequence repeats (SSRs) are prevalent in eukaryotic genome, especially in mammals. Repetitive sequences form approximately one-third of the camelid genomes, so study on this part of genome can be helpful in providing deeper information from the genome and its evolutionary path. Here, in order to improve our understanding regarding the camel genome architecture, the whole genome of the two dromedaries (Yazdi and Trodi camels) was sequenced. Totally, 92- and 84.3-Gb sequence data were obtained and assembled to 137,772 and 149,997 contigs with a N50 length of 54,626 and 54,031 bp in Yazdi and Trodi camels, respectively. Results showed that 30.58% of Yazdi camel genome and 30.50% of Trodi camel genome were covered by TEs. Contrary to the observed results in the genomes of cattle, sheep, horse, and pig, no endogenous retrovirus-K (ERVK) elements were found in the camel genome. Distribution pattern of DNA transposons in the genomes of dromedary, Bactrian, and cattle was similar in contrast with LINE, SINE, and long terminal repeat (LTR) families. Elements like RTE-BovB belonging to LINEs family in cattle and sheep genomes are dramatically higher than genome of dromedary. However, LINE1 (L1) and LINE2 (L2) elements cover higher percentage of LINE family in dromedary genome compared to genome of cattle. Also, 540,133 and 539,409 microsatellites were identified from the assembled contigs of Yazdi and Trodi dromedary camels, respectively. In both samples, di-(393,196) and tri-(65,313) nucleotide repeats contributed to about 42.5% of the microsatellites. The findings of the present study revealed that non-repetitive content of mammalian genomes is approximately similar. Results showed that 9.1 Mb (0.47% of whole assembled genome) of Iranian dromedary’s genome length is made up of SSRs. Annotation of repetitive content of Iranian dromedary camel genome revealed that 9,068 and 11,544 genes contain different types of TEs and SSRs, respectively. SSR markers identified in the present study can be used as a valuable resource for genetic diversity investigations and marker-assisted selection (MAS) in camel-breeding programs.

Emergence of Oxacillin Resistance in Stealth Methicillin-Resistant Staphylococcus aureus Due to mecA Sequence Instability.

Staphylococcus aureus strains that possess a mecA gene but are phenotypically susceptible to oxacillin and cefoxitin (OS-MRSA) have been recognized for over a decade and are a challenge for diagnostic laboratories. The mechanisms underlying the discrepancy vary from isolate to isolate. We characterized seven OS-MRSA clinical isolates of six different spa types from six different states by whole-genome sequencing to identify the nucleotide sequence changes leading to the OS-MRSA phenotype. The results demonstrated that oxacillin susceptibility was associated with mutations in regions of nucleotide repeats within mecA Subinhibitory antibiotic exposure selected for secondary mecA mutations that restored oxacillin resistance. Thus, strains of S. aureus that contain mecA but are phenotypically susceptible can become resistant after antibiotic exposure, which may result in treatment failure. OS-MRSA warrant follow-up susceptibility testing to ensure detection of resistant revertants.

Forensic characteristics and genetic analysis of both 27 Y-STRs and 143 Y-SNPs in Eastern Han Chinese population

Y-chromosome short tandem repeat (Y-STR) and Y-chromosome single nucleotide polymorphism (Y-SNP) frequency distributions provide resources for assessment of male population stratification among world-wide populations. Currently, the Y-STR Haplotype Reference Database (YHRD) contains numerous Y-chromosome haplotype profiles from various populations and countries around the world. However, for many of the recently discovered and already phylogenetically mapped Y-SNPs, the population data are scarce. Herein, the typing of 27 Y-STRs (Yfiler Plus) and 143 Y-SNPs (self-designed Y-SNP panel) was performed on 1269 unrelated males from 11 Han Chinese populations. Haplogroup O-M175 was the most predominant haplogroup in our Han Chinese data, ranging from 67.34% (Henan Han) to 93.16% (Guangdong Han). The highest haplogroup diversity (0.967056) was observed in Heilongjiang Han, with a discrimination capacity (DC) value of 0.3723. The number of alleles at single-copy loci varied from 2 for DYS391 (Guangdong Han) to 16 for DYS518 (Henan Han). For the majority of the populations (8/11), both the haplotype diversity and DC values are 1.0000. Furthermore, genetic differentiations were observed between Northern and Southern Han Chinese. These genetic differences were mainly reflected in haplogroup distribution and frequency, and they were confirmed by statistical analysis.

Mapping of scaffold/matrix attachment regions in human genome: a data mining exercise.

Scaffold/matrix attachment regions (S/MARs) are DNA elements that serve to compartmentalize the chromatin into structural and functional domains. These elements are involved in control of gene expression which governs the phenotype and also plays role in disease biology. Therefore, genome-wide understanding of these elements holds great therapeutic promise. Several attempts have been made toward identification of S/MARs in genomes of various organisms including human. However, a comprehensive genome-wide map of human S/MARs is yet not available. Toward this objective, ChIP-Seq data of 14 S/MAR binding proteins were analyzed and the binding site coordinates of these proteins were used to prepare a non-redundant S/MAR dataset of human genome. Along with co-ordinate (location) details of S/MARs, the dataset also revealed details of S/MAR features, namely, length, inter-SMAR length (the chromatin loop size), nucleotide repeats, motif abundance, chromosomal distribution and genomic context. S/MARs identified in present study and their subsequent analysis also suggests that these elements act as hotspots for integration of retroviruses. Therefore, these data will help toward better understanding of genome functioning and designing effective anti-viral therapeutics. In order to facilitate user friendly browsing and retrieval of the data obtained in present study, a web interface, MARome (http://bioinfo.net.in/MARome), has been developed.

Abstract 1741: Identification of microsatellite instability in hematologic malignant cell lines

Abstract Defective DNA repair machinery which is a consequence of chromosomal or molecular alterations is associated with variety of diseases including solid tumors. Cytogenetic analysis is an important tool in diagnosis and treatment of hematologic malignancies. Our aim is to understand the role of microsatellite instability in initiation and progression of blood cancers. Here we hypothesize that leukemogenic cells may carry defective repair mechanisms that contribute to other genomic instabilities. In order to explore the possible role of microsatellite instability (MSI) in leukemia and lymphoma, eight different DNA repair genes involved in mismatch repair (MMR) and direct reversal of damage (DRD) were analyzed in K562 (CML), Molt-4 (T-ALL) and Daudi (Burkitt’s lymphoma) cell lines. Fragment analysis was carried out using novel primers considering repeat motifs enriched with (A)n, (CA)n and (AT)n for instability. Almost all the cell lines showed moderate to high level of microsatellite instability and exhibited a shift in selected repeat regions indicating possible loss (66.7%) (MSH2, MSH4, MSH6, MLH1, MLH3, PMS1) or gain (25%) (MSH3, MGMT) of nucleotide repeats as compared to normal DNA. The K562, Philadelphia negative CML cell line appeared to be microsatellite stable (MSS) for genes MSH2 and MLH1. Treatment induced microsatellite instability is known in leukemic patient cells as also seen in Molt-4 cells. Our present and previous observations suggest that MSI is a non-random event in leukemia/lymphoma which needs confirmatory future studies in newly diagnosed untreated patient samples. Considering the fact that both MSI and MSS represents two distinct molecular pathways of carcinogenesis, assessment of MSI, if added as distinct diagnostic criteria, can possibly refine early detection and help in identifying novel targets in management of hematologic malignancies. Citation Format: Priyanjali Bhattacharya, Naveed Anjum Chikan, Arati Sharma, Dhimant Desai, Charyguly Annageldiyev, Pinaki Patel, Trupti N. Patel. Identification of microsatellite instability in hematologic malignant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1741.

Liquid-liquid phase separation and biomolecular condensates

Eukaryotic cells contain many membrane-less compartments, such as P granules, stress granules and p62 bodies, which are composed of highly concentrated biomacromolecules. These compartments are involved in various cellular processes, including germ cell specification, stress response, and selective autophagy. They are called “biomolecular condensates” and function in organizing cellular biochemical reactions, to make them relatively independent, highly ordered, and highly efficient. According to recent studies, biomolecular condensates exhibit liquid-like properties, such as a spherical shape, fusion upon contact and high dynamics. They arise from “liquid-liquid phase separation” (LLPS) driven by multivalent interactions among macromolecules. There are many ways to achieve macromolecular multivalence: Linear modular domains with similar function, like PRM repeats in N-WASP; oligomerization domains, like the PB1 domain in the selective autophagy receptor p62; similar post-translational modifications at multiple sites; poly-ubiquitination at one site; degenerate binding of RNA to proteins, etc. Additionally, intrinsically disordered regions (IDRs), sometimes called low complexity domains (LCDs), which contain high frequencies of a few specific amino acid types, are a common way to mediate multivalent interactions and drive LLPS. Many IDR-containing proteins, such as the P granule component PGL-3, the nuclear pore protein Nup98, and the RNA-binding protein FUS, were shown to undergo phase separation in vitro and localize to specific biomolecular condensates in vivo . RNAs with disease-related nucleotide repeat expansions, such as CUG repeats, CAG repeats, and GGGGCC repeats, can also form phase-separated puncta in vitro and in patients’ cells. Mechanisms for regulating phase separation include modulating the relationship between the concentration and saturation concentration of biomacromolecules, and altering biomolecular interactions. Regulation of transcription, translation and degradation can change biomacromolecule concentrations. Temperature, crowding agents, and ATP (which functions as a hydrotrope), can impact protein solubility. Protein modifications and novel interactions with other biomacromolecules can impact inter-molecular or/and intra-molecular interactions, thus disrupting phase separation. A special example is the nuclear transport receptor Kapβ2, which suppresses normal and aberrant phase separation of some RNA-binding proteins with IDRs, including FUS, by interacting with their PY-NLS region. The components that are essential for establishing phase separation are called “scaffolds”, and they enrich their “clients” via interaction through their free binding sites. The composition of clients can be altered by changing the stoichiometries of scaffolds. Droplets formed by different biomacromolecules, such as FIB1 and NPM, can co-exist to form immiscible multi-phase condensates, which are analogous to multi-layered membrane-less organelles in vivo , such as nucleoli and stress granules. Many in vitro phase-separation systems have been established, which can induce receptor clustering, activate actin or tubulin polymerization, facilitate enzymatic reactions, etc. Some studies have suggested a link between phase separation and heterochromatin formation or gene control. Based on reconstituted phase separation systems in vitro , and their links to biomolecular condensates in vivo , we can gain deep understanding of the assembly, dissolution, composition, physical properties, biochemical activities and cellular functions of biomolecular condensates.

Genome survey and SSR analysis of Apocynum venetum.

Apocynum venetum is an eco-economic plant that exhibits high stress resistance. In the present paper, we carried out a whole-genome survey of A. venetum in order to provide a foundation for its whole-genome sequencing. High-throughput sequencing technology (Illumina NovaSep) was first used to measure the genome size of A. venetum, and bioinformatics methods were employed for the evaluation of the genome size, heterozygosity ratio, repeated sequences, and GC content in order to provide a foundation for subsequent whole-genome sequencing. The sequencing analysis results indicated that the preliminary estimated genome size of A. venetum was 254.40 Mbp, and its heterozygosity ratio and percentage of repeated sequences were 0.63 and 40.87%, respectively, indicating that it has a complex genome. We used k-mer = 41 to carry out a preliminary assembly and obtained contig N50, which was 3841 bp with a total length of 223949699 bp. We carried out further assembly to obtain scaffold N50, which was 6196 bp with a total length of 227322054 bp. We performed simple sequence repeat (SSR) molecular marker prediction based on the A. venetum genome data and identified a total of 101918 SSRs. The differences between the different types of nucleotide repeats were large, with mononucleotide repeats being most numerous and hexanucleotide repeats being least numerous. We recommend the use of the ‘2+3’ (Illumina+PacBio) sequencing combination to supplement the Hi-C technique and resequencing technique in future whole-genome research in A. venetum.

WC-1 and the Proximal GATA Sequence Mediate a Cis-/Trans-Acting Repressive Regulation of Light-Dependent Gene Transcription in the Dark.

Simple SummaryWe observed that both the proximal GATA sequence in light-responsive elements (LREs) of the albino-3 promoter and the Zinc Finger Domain of WC-1 are involved in the dark-related repressive control mechanism of light-regulated genes.Light influences a wide range of physiological processes from prokaryotes to mammals. Neurospora crassa represents an important model system used for studying this signal pathway. At molecular levels, the WHITE COLLAR Complex (WCC), a heterodimer formed by WC-1 (the blue light photo-sensor) and WC-2 (the transcriptional activator), is the critical positive regulator of light-dependent gene expression. GATN (N indicates any other nucleotide) repeats are consensus sequences within the promoters of light-dependent genes recognized by the WCC. The distal GATN is also known as C-box since it is involved in the circadian clock. However, we know very little about the role of the proximal GATN, and the molecular mechanism that controls the transcription of light-induced genes during the dark/light transition it is still unclear. Here we showed a first indication that mutagenesis of the proximal GATA sequence within the target promoter of the albino-3 gene or deletion of the WC-1 zinc finger domain led to a rise in expression of light-dependent genes already in the dark, effectively decoupling light stimuli and transcriptional activation. This is the first observation of cis-/trans-acting repressive machinery, which is not consistent with the light-dependent regulatory mechanism observed in the eukaryotic world so far.

Frameshift Mutations and Loss of Expression of CLCA4 Gene are Frequent in Colorectal Cancers With Microsatellite Instability.

Chloride channel calcium-activated (CLCA) genes encode regulators for chloride transport across the cell membrane. As for cancer development, some CLCA genes are considered putative tumor suppressor genes. The aim of this study was to explore whether CLCA4 gene would have mutations in its nucleotide repeats in colorectal cancer (CRC). In a public database, we found that CLCA4 gene had mononucleotide repeats in the coding sequences that might be mutational targets in the cancers with microsatellite instability. For this, the current study studied 146 CRCs for mutation and expression analyses by single-strand conformation polymorphism analysis, DNA sequencing, and immunohistochemistry. Overall, we found CLCA4 frameshift mutations in 12/101 (11.8%) CRCs with high-microsatellite instability (MSI-H), but none in microsatellite stable CRCs (0/45) (P<0.01). In addition, we analyzed intratumoral heterogeneity of the CLCA4 frameshift mutations and found that 1 CRC harbored regional intratumoral heterogeneity of the CLCA4 frameshift mutation. Loss of CLCA4 protein expression was identified in 50% of CRCs. Also, cancers with MSI-H harboring CLCA4 frameshift mutations showed lower CLCA4 immunostaining than those with the wild-type. Our data indicate that the CLCA4 gene harbors alterations both in somatic mutation and expression, suggesting their roles in tumorigenesis of CRC with MSI-H.

Genetic dissociation of three antigenic genes in Plasmodium ovale curtisi and Plasmodium ovale wallikeri

Plasmodium ovale curtisi and Plasmodium ovale wallikeri are two sympatric human malaria species prevalent in Africa, Asia and Oceania. The reported prevalence of both P. ovale spp. was relatively low compared to other malaria species, but more sensitive molecular detection techniques have shown that asymptomatic low-density infections are more common than previously thought. Whole genome sequencing of both P. ovale spp. revealed genetic dissociation between P. ovale curtisi and P. ovale wallikeri suggesting a species barrier. In this study we further evaluate such a barrier by assessing polymorphisms in the genes of three vaccine candidate surface protein: circumsporozoite protein/ thrombospondin-related anonymous-related protein (ctrp), circumsporozoite surface protein (csp) and merozoite surface protein 1 (msp1). The complete coding sequence of ctrp and csp, and a partial fragment of msp1 were isolated from 25 P. ovale isolates and compared to previously reported reference sequences. A low level of nucleotide diversity (Pi = 0.02–0.10) was observed in all three genes. Various sizes of tandem repeats were observed in all ctrp, csp and msp1 genes. Both tandem repeat unit and nucleotide polymorphism in all three genes exhibited clear dimorphism between P. ovale curtisi and P. ovale wallikeri, supporting evidence of non-recombination between these two species.

Assessment of genome stability in various breeds of cattle.

Chromosomal instability is a type of genome instability involving changes in genetic information at the chromosomal level. The basic tests used to identify this form of instability are sister chromatid exchange (SCE) tests and identification of fragile sites (FS). SCE is the process by which sister chromatids become fragmented as a result of DNA strand breakage and reassembly, followed by exchange of these fragments. FS can be observed in the form of breaks, gaps or constrictions on chromosomes, which often result from multiple nucleotide repeats in DNA that are difficult to replicate. The research material was the peripheral blood of ten breeds of cattle raised in Poland, including four native breeds covered by a genetic resources conservation programme, i.e. Polish Red, Polish Red-and-White, White-Backed, and Polish Black-and-White, as well as Polish Holstein-Friesian, Simmental, Montbéliarde, Jersey, Limousine and Danish Red. Two tests were performed on chromosomes obtained from in vitro cultures: SCE and FS. The average frequency of SCE was 5.08 ± 1.31, while the incidence of FS was 3.45 ± 0.94. Differences in the incidence of SCE and FS were observed between breeds. The least damage was observed in the Polish Red and White-Backed breeds, and the most in Polish Holstein-Friesians. The most damage was observed in the interstitial part of the chromosomes. Age was shown to significantly affect the incidence of SCE and FS. Younger cows showed less damage than older ones (SCE: 4.84 ± 1.25; 5.34 ± 1.24; FS: 3.10 ± 0.88, 3.80 ± 0.92).

Short tandem repeats and methylation in the promoter region affect expression of cystathionine beta-synthase gene in the laying hen

Homocysteine can be converted to cysteine via the transsulfuration pathway where cystathionine beta-synthase (CBS) is a rate-limiting enzyme. Homocysteine is thought to play a role in bone remodelling and strength. Previous results indicated that some of the difference in bone strength of end-of-lay hens may be associated with CBS expression level. To investigate if differences in the promoter region of the CBS gene might be responsible for observed differences in gene expression between CBS alleles post mortem- and in-vitro expression studies have been undertaken. Transfection of the DF-1 avian cell line with a series of deletion fragments of the 5′ promoter, or constructs containing three CBS allele sequences, with a luciferase reporter revealed that a core region of −155 to +131 bp in the CBS promoter was essential for mRNA expression. We found that a variable number of short tandem repeats (7 nucleotide motif and 6 nucleotide repeats) in the core region of the promoter affecting the transcriptional activity and a strong effect for gene expression. However, methylation of the 6 nucleotide repeats varied between allelic variants and these maybe responsible for differences in promoter activity. Our findings indicated variable short tandem repeats and the differentially methylated sites in the promoter region may be responsible for CBS expression differences in the bone of laying hens.

In silico identification of long non-coding RNA based simple sequence repeat markers and their application in diversity analysis in rice

In rice (Oryza sativa L.), there are only few simple sequence repeat (SSR) markers that have been developed from the non-coding regions of the genome. Hence, a genome-wide survey of simple sequence repeats (SSR) marker was conducted in the newly discovered 2225 long non-coding RNA (lnc-RNA) genes of rice, which resulted in 937 SSR markers, of which 644 markers contain 7 or more repeat nucleotides. Di-nucleotide repeat motifs were found highest in number followed by tri-nucleotide repeats. The result shows that repeat length has an inverse relationship to its number. Validation of 22 SSR markers, which distributed in 12 independent chromosomes, was performed in 24 diverse rice genotypes. The exotic materials were found to be genetically more diverse than the indigenous landraces. The indigenous landraces and exotic materials have shown the polymorphic information content values of 0.21 and 0.39, respectively, which supports their genetic diversity status. Thus, the results illustrate the lnc-RNA based SSR marker system as an effective, novel source for the analysis of genetic diversity or genotyping in rice.

Minimal microsatellite shift in microsatellite instability high endometrial cancer: a significant pitfall in diagnostic interpretation

Mismatch-repair deficiency testing plays a critical role in the identification of proband in Lynch Syndrome families and triaging patients with high stage or recurrent solid malignancies for check point inhibitor (Pembrolizumab) immunotherapy. We compared microsatellite shift patterns of microsatellite instability PCR analysis at 5 NCI recommended loci between microsatellite instability high endometrial carcinoma (n = 50) and microsatellite instability high colorectal cancer (n = 19). The endometrial cancer cohort included 45 endometrioid, 1 serous, and 4 clear cell carcinomas. Overall, 52% (26/50) of microsatellite instability high endometrial cancers showed minimal microsatellite shift (defined as a one to three nucleotide repeat shift at an involved locus) observed at least at one locus. Among microsatellite instability high endometrial cancers with minimal microsatellite shift, the frequencies at each involved locus were D2S123 (21/21, 100%), D17S250 (10/11, 89%), D5S346 (11/12, 92%), BAT25 (9/12, 80%), and BAT26 (8/21, 45%). Noticeably, 11 of the 26 cases (42%) showed only minimal shift. Among microsatellite instability high endometrial cancers with minimal microsatellite shift, 65% (17/26) had combined MLH1 and PMS2 loss, 8% (2/26) had combined MSH2 and MSH6 loss, 13% (3/26) had MSH6 loss and 15% (4/26) had loss of PMS2 by immunohistochemistry. In contrast, only 16% (3/19) had minimal microsatellite shift seen in colorectal cancer cohort with corresponding loss of MLH1/PMS2, MSH2/MSH6, or MSH6. Overall, 15% (7/50) of microsatellite instability high endometrial carcinomas showed isolated loss of MSH6 in contrast to 7% (1/15) seen in microsatellite instability high colorectal carcinomas. In conclusion, microsatellite instability high endometrial carcinomas have a significantly higher frequency of minimal microsatellite shift that coincides with a high percentage of combined loss of MLH1/PMS2. Microsatellite instability high endometrial cancers also have more frequent loss of MSH-6. Diagnostically, recognition of minimal microsatellite shift is crucial for accurate interpretation of microsatellite instability PCR data of endometrial carcinoma.