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Fulltext Limited Polymorphism in the Dihydrofolate Reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand

Sangsri R, Choowongkomon K, Tuntipaiboontana R, Sugaram R, Boondej P, Sudathip P, et al.

Acta Trop, 2023 Dec;248:107016.
PMID: 37683820 DOI: 10.1016/j.actatropica.2023.107016

BACKGROUND: The 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of Disease Control in Thailand, suggesting a persistent infection. Little is known about dihydrofolate reductase (pkdhfr) and dihydropteroate synthase (pkdhps), putative antimalarial resistance markers for P. knowlesi. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating pkdhps mutations. The haplotype pattern of pkdhfr-pkdhps in Thai isolates was analyzed, and the effects of these pkdhps mutations were predicted by using a computer program.
METHODS: Pkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein-ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates.
RESULTS: Five nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein-ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr-pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates.
CONCLUSIONS: A new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.

Matched MeSH terms: Dihydropteroate Synthase/genetics; Drug Resistance/genetics; Plasmodium falciparum/genetics; Tetrahydrofolate Dehydrogenase/genetics
Fulltext NKX3.1 Expression and Molecular Characterization of Secretory Myoepithelial Carcinoma (SMCA): Advancing the Case for a Salivary Mucous Acinar Phenotype

Patel S, Wald AI, Bastaki JM, Chiosea SI, Singhi AD, Seethala RR

Head Neck Pathol, 2023 Jun;17(2):467-478.
PMID: 36746884 DOI: 10.1007/s12105-023-01524-2

BACKGROUND: Secretory myoepithelial carcinomas (SMCA) are rare, mucinous, signet ring predominant tumors with primitive myoepithelial features. While many mucinous salivary gland tumors have now been molecularly characterized, key drivers in SMCA have yet to be elucidated. Recently, NKX3.1, a homeodomain transcription factor implicated in salivary mucous acinar development was also shown in a subset of salivary mucinous neoplasms, salivary intraductal papillary mucinous neoplasms (SG-IPMN). To date, NKX3.1 expression has not been characterized in other mucinous salivary lesions. Here, we report molecular and extended immunophenotypic findings in SMCA and NKX3.1 expression in the context of other head and neck lesions.
METHODS: We retrieved 4 previously reported SMCA, performed additional immunohistochemical and targeted next-generation sequencing (NGS). We also investigated the use of NKX3.1 as a marker for SMCA in the context of its prevalence and extent (using H-score) in a mixed cohort of retrospectively and prospectively tested head and neck lesions (n = 223) and non-neoplastic tissues (n = 66).
RESULTS: NKX3.1 positivity was confirmed in normal mucous acini as well as in mucous acinar class of lesions (5/6, mean H-score: 136.7), including mucinous adenocarcinomas (3/4), SG-IPMN (1/1), and microsecretory adenocarcinoma (MSA) (1/1). All SMCA were positive. Fluorescence in situ hybridization for SS18 rearrangements were negative in all successfully tested cases (0/3). NGS was successful in two cases (cases 3 and 4). Case 3 demonstrated a PTEN c.655C>T p.Q219* mutation and a SEC16A::NOTCH1 fusion while case 4 (clinically aggressive) showed a PTEN c.1026+1G>A p.K342 splice site variant, aTP53 c.524G>A p.R175H mutation and a higher tumor mutation burden (29 per Mb). PTEN immunohistochemical loss was confirmed in both cases and a subset of tumor cells showed strong (extreme) staining for P53 in Case 4.
CONCLUSION: Despite a partial myoepithelial phenotype, SMCA, along with mucinous adenocarcinomas/SG-IPMN and MSA, provisionally constitute a mucous acinar class of tumors based on morphology and NKX3.1 expression. Like salivary mucinous adenocarcinomas/SG-IPMN, SMCA also show alterations of the PTEN/PI3K/AKT pathway and may show progressive molecular alterations. We document the first extramammary tumor with a SEC16A::NOTCH1 fusion.

Matched MeSH terms: Transcription Factors/genetics; Biomarkers, Tumor/genetics; Phosphatidylinositol 3-Kinases/genetics; Vesicular Transport Proteins/genetics
Controlled expression of cholera toxin B subunit from Vibrio cholerae in Escherichia coli

Haryanti T, Mariana NS, Latifah SY, Yusoff K, Raha AR

Pak J Biol Sci, 2008 Jul 01;11(13):1718-22.
PMID: 18819625

The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.

Matched MeSH terms: Cholera Toxin/genetics; Escherichia coli/genetics; Genetic Vectors/genetics; Vibrio cholerae/genetics
Fulltext Characterizing prostate cancer risk through multi-ancestry genome-wide discovery of 187 novel risk variants

Wang A, Shen J, Rodriguez AA, Saunders EJ, Chen F, Janivara R, et al.

Nat Genet, 2023 Dec;55(12):2065-2074.
PMID: 37945903 DOI: 10.1038/s41588-023-01534-4

The transferability and clinical value of genetic risk scores (GRSs) across populations remain limited due to an imbalance in genetic studies across ancestrally diverse populations. Here we conducted a multi-ancestry genome-wide association study of 156,319 prostate cancer cases and 788,443 controls of European, African, Asian and Hispanic men, reflecting a 57% increase in the number of non-European cases over previous prostate cancer genome-wide association studies. We identified 187 novel risk variants for prostate cancer, increasing the total number of risk variants to 451. An externally replicated multi-ancestry GRS was associated with risk that ranged from 1.8 (per standard deviation) in African ancestry men to 2.2 in European ancestry men. The GRS was associated with a greater risk of aggressive versus non-aggressive disease in men of African ancestry (P = 0.03). Our study presents novel prostate cancer susceptibility loci and a GRS with effective risk stratification across ancestry groups.

Matched MeSH terms: Hispanic Americans/genetics; African Continental Ancestry Group/genetics; European Continental Ancestry Group/genetics; Asian Continental Ancestry Group/genetics
Fulltext Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement

Kaiyrzhanov R, Mohammed SEM, Maroofian R, Husain RA, Catania A, Torraco A, et al.

Am J Hum Genet, 2022 Sep 01;109(9):1692-1712.
PMID: 36055214 DOI: 10.1016/j.ajhg.2022.07.007

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

Matched MeSH terms: Homeostasis/genetics; Membrane Proteins/genetics; Mitochondria/genetics; Mitochondrial Proteins/genetics
Curcumin inhibits prostate cancer by upregulating miR-483-3p and inhibiting UBE2C

Li W, Wang F, Wang X, Xu W, Liu F, Hu R, et al.

J Biochem Mol Toxicol, 2024 Feb;38(2):e23645.
PMID: 38348716 DOI: 10.1002/jbt.23645

Prostate cancer (PCa) is an extremely common genitourinary malignancy among elderly men. Many evidence have shown the efficacy of curcumin (CUR) in inhibiting the progression of PCa. However, the pharmacological function of CUR in PCa is still not quite clear. In this research, CUR was found to suppress the proliferation and enhance the apoptotic rate in in vitro PCa cell models in a dose- and time-dependent manner. In a xenograft animal model, the administration of CUR contributed to a significant decrease in the growth of the xenograft tumor induced by the transplanted PC-3 cells. Ubiquitin-conjugating enzyme E2 C is implicated in the modulation of multiple types of cancers. In humans, the expression levels of UBE2C are significantly higher in PCa versus benign prostatic hyperplasia. Treatment with CUR decreased the expression of UBE2C, whereas it increased miR-483-3p expression. In contrast with the control mice, the CUR-treated mice showed a significant reduction in UBE2C and Ki-67 in PCa cells. The capability of proliferation, migration, and invasion of PCa cells was inhibited by the knockdown of UBE2C mediated by siRNA. Furthermore, dual luciferase reporter gene assay indicated the binding of miR-483-3p to UBE2C. In summary, CUR exerts its antitumor effects through regulation of the miR-483-3p/UBE2C axis by decreasing UBE2C and increasing miR-483-3p. The findings may also provide new molecular markers for PCa diagnosis and treatment.

Matched MeSH terms: Cell Movement/genetics; Apoptosis/genetics; Ubiquitin-Conjugating Enzymes/genetics; Cell Proliferation/genetics
Fulltext Dimensionality reduction reveals fine-scale structure in the Japanese population with consequences for polygenic risk prediction

Sakaue S, Hirata J, Kanai M, Suzuki K, Akiyama M, Lai Too C, et al.

Nat Commun, 2020 03 26;11(1):1569.
PMID: 32218440 DOI: 10.1038/s41467-020-15194-z

The diversity in our genome is crucial to understanding the demographic history of worldwide populations. However, we have yet to know whether subtle genetic differences within a population can be disentangled, or whether they have an impact on complex traits. Here we apply dimensionality reduction methods (PCA, t-SNE, PCA-t-SNE, UMAP, and PCA-UMAP) to biobank-derived genomic data of a Japanese population (n = 169,719). Dimensionality reduction reveals fine-scale population structure, conspicuously differentiating adjacent insular subpopulations. We further enluciate the demographic landscape of these Japanese subpopulations using population genetics analyses. Finally, we perform phenome-wide polygenic risk score (PRS) analyses on 67 complex traits. Differences in PRS between the deconvoluted subpopulations are not always concordant with those in the observed phenotypes, suggesting that the PRS differences might reflect biases from the uncorrected structure, in a trait-dependent manner. This study suggests that such an uncorrected structure can be a potential pitfall in the clinical application of PRS.

Matched MeSH terms: Genetics, Population*; Multifactorial Inheritance/genetics*; Asian Continental Ancestry Group/genetics*
Fulltext Identification of potential Campylobacter jejuni genes involved in biofilm formation by EZ-Tn5 Transposome mutagenesis

Teh AHT, Lee SM, Dykes GA

BMC Res Notes, 2017 May 12;10(1):182.
PMID: 28499399 DOI: 10.1186/s13104-017-2504-1

BACKGROUND: Biofilm formation has been suggested to play a role in the survival of Campylobacter jejuni in the environment and contribute to the high incidence of human campylobacteriosis. Molecular studies of biofilm formation by Campylobacter are sparse.
RESULTS: We attempted to identify genes that may be involved in biofilm formation in seven C. jejuni strains through construction of mutants using the EZ-Tn5 Transposome system. Only 14 mutants with reduced biofilm formation were obtained, all from one strain of C. jejuni. Three different genes of interest, namely CmeB (synthesis of multidrug efflux system transporter proteins), NusG (transcription termination and anti-termination protein) and a putative transmembrane protein (involved in membrane protein function) were identified. The efficiency of the EZ::TN5 transposon mutagenesis approach was strain dependent and was unable to generate any mutants from most of the strains used.
CONCLUSIONS: A diverse range of genes may be involved in biofilm formation by C. jejuni. The application of the EZ::TN5 system for construction of mutants in different Campylobacter strains is limited.

Matched MeSH terms: Bacterial Proteins/genetics; DNA Transposable Elements/genetics*; Genes, Bacterial/genetics*; Campylobacter jejuni/genetics*
Fulltext Genetic diversity of Pantoea stewartii subspecies stewartii causing jackfruit-bronzing disease in Malaysia

Abidin N, Ismail SI, Vadamalai G, Yusof MT, Hakiman M, Karam DS, et al.

PLoS One, 2020;15(6):e0234350.
PMID: 32530926 DOI: 10.1371/journal.pone.0234350

Jackfruit-bronzing is caused by bacteria Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii), showing symptoms of yellowish-orange to reddish discolouration and rusty specks on pulps and rags of jackfruit. Twenty-eight pure bacterial strains were collected from four different jackfruit outbreak collection areas in Peninsular Malaysia (Jenderam, Maran, Muadzam Shah and Ipoh). Positive P. stewartii subsp. stewartii verification obtained in the study was based on the phenotypic, hypersensitivity, pathogenicity and molecular tests. Multilocus sequence analysis (MLSA) was performed using four housekeeping genes (gyrB, rpoB, atpD and infB) on all 28 bacterial strains. Single gyrB, rpoB, atpD and infB phylogenetic trees analyses revealed the bootstrap value of 99-100% between our bacterial strains with P. stewartii subsp. stewartii reference strains and P. stewartii subsp. indologenes reference strains. On the other hand, phylogenetic tree of the concatenated sequences of the four housekeeping genes revealed that our 28 bacterial strains were more closely related to P. stewartii subsp. stewartii (99% similarities) compared to its close relative P. stewartii subsp. indologenes, although sequence similarity between these two subspecies were up to 100%. All the strains collected from the four collection areas clustered together, pointing to no variation among the bacterial strains. This study improves our understanding and provided new insight on the genetic diversity of P. stewartii subsp. stewartii associated with jackfruit-bronzing in Malaysia.

Matched MeSH terms: DNA, Bacterial/genetics; Virulence/genetics; Pantoea/genetics*; DNA, Intergenic/genetics
Fulltext Assessment of four molecular markers as potential DNA barcodes for red algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta)

Tan J, Lim PE, Phang SM, Hong DD, Sunarpi H, Hurtado AQ

PLoS One, 2012;7(12):e52905.
PMID: 23285223 DOI: 10.1371/journal.pone.0052905

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.

Matched MeSH terms: Rhodophyta/genetics*; DNA, Mitochondrial/genetics; DNA, Plant/genetics; DNA, Intergenic/genetics
Fulltext Comparison of eight complete chloroplast genomes of the endangered Aquilaria tree species (Thymelaeaceae) and their phylogenetic relationships

Hishamuddin MS, Lee SY, Ng WL, Ramlee SI, Lamasudin DU, Mohamed R

Sci Rep, 2020 Aug 03;10(1):13034.
PMID: 32747724 DOI: 10.1038/s41598-020-70030-0

Aquilaria tree species are naturally distributed in the Indomalesian region and are protected against over-exploitation. They produce a fragrant non-timber product of high economic value, agarwood. Ambiguous species delimitation and limited genetic information within Aquilaria are among the impediments to conservation efforts. In this study, we conducted comparative analysis on eight Aquilaria species complete chloroplast (cp) genomes, of which seven were newly sequenced using Illumina HiSeq X Ten platform followed by de novo assembly. Aquilaria cp genomes possess a typical quadripartite structure including gene order and genomic structure. The length of each of the cp genome is about 174 kbp and encoded between 89 and 92 proteins, 38 tRNAs, and 8 rRNAs, with 27 duplicated in the IR (inverted repeat) region. Besides, 832 repeats (forward, reverse, palindrome and complement repeats) and nine highly variable regions were also identified. The phylogenetic analysis suggests that the topology structure of Aquilaria cp genomes were well presented with strong support values based on the cp genomes data set and matches their geographic distribution pattern. In summary, the complete cp genomes will facilitate development of species-specific molecular tools to discriminate Aquilaria species and resolve the evolutionary relationships of members of the Thymelaeaceae family.

Matched MeSH terms: Base Composition/genetics; Nucleotides/genetics; Thymelaeaceae/genetics*; Inverted Repeat Sequences/genetics
Fulltext Spatially Enriched Paralog Rearrangements Argue Functionally Diverse Ribosomes Arise during Cold Acclimation in Arabidopsis

Martinez-Seidel F, Beine-Golovchuk O, Hsieh YC, Eshraky KE, Gorka M, Cheong BE, et al.

Int J Mol Sci, 2021 Jun 07;22(11).
PMID: 34200446 DOI: 10.3390/ijms22116160

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.

Matched MeSH terms: Ribosomal Proteins/genetics; Ribosomes/genetics; Arabidopsis/genetics; Arabidopsis Proteins/genetics
Fulltext TRANSPARENT TESTA GLABRA1 Regulates the Accumulation of Seed Storage Reserves in Arabidopsis

Chen M, Zhang B, Li C, Kulaveerasingam H, Chew FT, Yu H

Plant Physiol, 2015 Sep;169(1):391-402.
PMID: 26152712 DOI: 10.1104/pp.15.00943

Seed storage reserves mainly consist of starch, triacylglycerols, and storage proteins. They not only provide energy for seed germination and seedling establishment, but also supply essential dietary nutrients for human beings and animals. So far, the regulatory networks that govern the accumulation of seed storage reserves in plants are still largely unknown. Here, we show that TRANSPARENT TESTA GLABRA1 (TTG1), which encodes a WD40 repeat transcription factor involved in many aspects of plant development, plays an important role in mediating the accumulation of seed storage reserves in Arabidopsis (Arabidopsis thaliana). The dry weight of ttg1-1 embryos significantly increases compared with that of wild-type embryos, which is accompanied by an increase in the contents of starch, total protein, and fatty acids in ttg1-1 seeds. FUSCA3 (FUS3), a master regulator of seed maturation, binds directly to the TTG1 genomic region and suppresses TTG1 expression in developing seeds. TTG1 negatively regulates the accumulation of seed storage proteins partially through transcriptional repression of 2S3, a gene encoding a 2S albumin precursor. TTG1 also indirectly suppresses the expression of genes involved in either seed development or synthesis/modification of fatty acids in developing seeds. In addition, we demonstrate that the maternal allele of the TTG1 gene suppresses the accumulation of storage proteins and fatty acids in seeds. Our results suggest that TTG1 is a direct target of FUS3 in the framework of the regulatory hierarchy controlling seed filling and regulates the accumulation of seed storage proteins and fatty acids during the seed maturation process.

Matched MeSH terms: Mutation/genetics; Seeds/genetics; Arabidopsis/genetics; Arabidopsis Proteins/genetics
Fulltext Genetic diversity of Eurycoma longifolia inferred from single nucleotide polymorphisms

Osman A, Jordan B, Lessard PA, Muhammad N, Haron MR, Riffin NM, et al.

Plant Physiol, 2003 Mar;131(3):1294-301.
PMID: 12644679 DOI: 10.1104/pp.012492

Eurycoma longifolia Jack. is a treelet that grows in the forests of Southeast Asia and is widely used throughout the region because of its reported medicinal properties. Widespread harvesting of wild-grown trees has led to rapid thinning of natural populations, causing a potential decrease in genetic diversity among E. longifolia. Suitable genetic markers would be very useful for propagation and breeding programs to support conservation of this species, although no such markers currently exist. To meet this need, we have applied a genome complexity reduction strategy to identify a series of single nucleotide polymorphisms (SNPs) within the genomes of several E. longifolia accessions. We have found that the occurrence of these SNPs reflects the geographic origins of individual plants and can distinguish different natural populations. This work demonstrates the rapid development of molecular genetic markers in species for which little or no genomic sequence information is available. The SNP markers that we have developed in this study will also be useful for identifying genetic fingerprints that correlate with other properties of E. longifolia, such as high regenerability or the appearance of bioactive metabolites.

Matched MeSH terms: Genetic Markers/genetics; Plants, Medicinal/genetics; Polymorphism, Single Nucleotide/genetics*; Eurycoma/genetics*
Fulltext A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi

Divis PC, Shokoples SE, Singh B, Yanow SK

Malar J, 2010 Nov 30;9:344.
PMID: 21114872 DOI: 10.1186/1475-2875-9-344

BACKGROUND: The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples.
METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.

Matched MeSH terms: DNA, Ribosomal/genetics; DNA, Protozoan/genetics; Plasmodium knowlesi/genetics; DNA Primers/genetics
Fulltext Multiple-locus variable-number tandem repeat analysis of Vibrio cholerae in comparison with pulsed field gel electrophoresis and virulotyping

Teh CS, Chua KH, Thong KL

J Biomed Biotechnol, 2010;2010:817190.
PMID: 20671932 DOI: 10.1155/2010/817190

Molecular analysis of Malaysian Vibrio cholerae was carried out using a multiple-locus variable-number tandem repeat analysis (MLVA) assay based on 7 loci of V. cholerae. The discriminatory ability of the assay was compared with pulsed-field gel electrophoresis (PFGE) using 43 Malaysian V. cholerae isolated from various sources. In addition, the virulotypes of the strains were determined. Based on MLVA, 38 allelic profiles were obtained (F = 0.63) while PFGE generated 35 pulsotypes (F = 0.71). Simpson's index of diversity for different VNTR loci ranged from 0.59 to 0.92. The combined loci increased the discriminatory index to 0.99 which was comparable with PFGE (D = 0.99). Most of the environmental non-O1/non-O139 strains harbored rtxA, rstR, toxR, and hlyA only, and the virulotype of this serogroup was significantly different (P < .01) from clinical/environmental O1 and environmental O139 strains. In conclusion, the MLVA assay developed in this study was a useful genotyping tool with comparable discriminatory power with PFGE. In addition, the combination of the two approaches can further distinguish the strains from different sources and geographical regions of isolation.

Matched MeSH terms: Vibrio cholerae/genetics; Virulence/genetics; Minisatellite Repeats/genetics*; Genetic Loci/genetics*
Fulltext Finding the factors of reduced genetic diversity on X chromosomes of Macaca fascicularis: male-driven evolution, demography, and natural selection

Osada N, Nakagome S, Mano S, Kameoka Y, Takahashi I, Terao K

Genetics, 2013 Nov;195(3):1027-35.
PMID: 24026095 DOI: 10.1534/genetics.113.156703

The ratio of genetic diversity on X chromosomes relative to autosomes in organisms with XX/XY sex chromosomes could provide fundamental insight into the process of genome evolution. Here we report this ratio for 24 cynomolgus monkeys (Macaca fascicularis) originating in Indonesia, Malaysia, and the Philippines. The average X/A diversity ratios in these samples was 0.34 and 0.20 in the Indonesian-Malaysian and Philippine populations, respectively, considerably lower than the null expectation of 0.75. A Philippine population supposed to derive from an ancestral population by founding events showed a significantly lower ratio than the parental population, suggesting a demographic effect for the reduction. Taking sex-specific mutation rate bias and demographic effect into account, expected X/A diversity ratios generated by computer simulations roughly agreed with the observed data in the intergenic regions. In contrast, silent sites in genic regions on X chromosomes showed strong reduction in genetic diversity and the observed X/A diversity ratio in the genic regions cannot be explained by mutation rate bias and demography, indicating that natural selection also reduces the level of polymorphism near genes. Whole-genome analysis of a female cynomolgus monkey also supported the notion of stronger reduction of genetic diversity near genes on the X chromosome.

Matched MeSH terms: DNA/genetics; Genetics, Population; Macaca fascicularis/genetics*; X Chromosome/genetics*
Fulltext Molecular characterization of α- and β-thalassaemia among Malay patients

Yatim NF, Rahim MA, Menon K, Al-Hassan FM, Ahmad R, Manocha AB, et al.

Int J Mol Sci, 2014 May 19;15(5):8835-45.
PMID: 24857915 DOI: 10.3390/ijms15058835

Both α- and β-thalassaemia syndromes are public health problems in the multi-ethnic population of Malaysia. To molecularly characterise the α- and β-thalassaemia deletions and mutations among Malays from Penang, Gap-PCR and multiplexed amplification refractory mutation systems were used to study 13 α-thalassaemia determinants and 20 β-thalassaemia mutations in 28 and 40 unrelated Malays, respectively. Four α-thalassaemia deletions and mutations were demonstrated. --SEA deletion and αCSα accounted for more than 70% of the α-thalassaemia alleles. Out of the 20 β-thalassaemia alleles studied, nine different β-thalassaemia mutations were identified of which βE accounted for more than 40%. We concluded that the highest prevalence of (α- and β-thalassaemia alleles in the Malays from Penang are --SEA deletion and βE mutation, respectively.

Matched MeSH terms: alpha-Thalassemia/genetics*; beta-Thalassemia/genetics*; Asian Continental Ancestry Group/genetics*; alpha-Globins/genetics
Fulltext Analysis of the leaf transcriptome of Musa acuminata during interaction with Mycosphaerella musicola: gene assembly, annotation and marker development

Passos MA, de Cruz VO, Emediato FL, de Teixeira CC, Azevedo VC, Brasileiro AC, et al.

BMC Genomics, 2013 Feb 05;14:78.
PMID: 23379821 DOI: 10.1186/1471-2164-14-78

BACKGROUND: Although banana (Musa sp.) is an important edible crop, contributing towards poverty alleviation and food security, limited transcriptome datasets are available for use in accelerated molecular-based breeding in this genus. 454 GS-FLX Titanium technology was employed to determine the sequence of gene transcripts in genotypes of Musa acuminata ssp. burmannicoides Calcutta 4 and M. acuminata subgroup Cavendish cv. Grande Naine, contrasting in resistance to the fungal pathogen Mycosphaerella musicola, causal organism of Sigatoka leaf spot disease. To enrich for transcripts under biotic stress responses, full length-enriched cDNA libraries were prepared from whole plant leaf materials, both uninfected and artificially challenged with pathogen conidiospores.
RESULTS: The study generated 846,762 high quality sequence reads, with an average length of 334 bp and totalling 283 Mbp. De novo assembly generated 36,384 and 35,269 unigene sequences for M. acuminata Calcutta 4 and Cavendish Grande Naine, respectively. A total of 64.4% of the unigenes were annotated through Basic Local Alignment Search Tool (BLAST) similarity analyses against public databases.Assembled sequences were functionally mapped to Gene Ontology (GO) terms, with unigene functions covering a diverse range of molecular functions, biological processes and cellular components. Genes from a number of defense-related pathways were observed in transcripts from each cDNA library. Over 99% of contig unigenes mapped to exon regions in the reference M. acuminata DH Pahang whole genome sequence. A total of 4068 genic-SSR loci were identified in Calcutta 4 and 4095 in Cavendish Grande Naine. A subset of 95 potential defense-related gene-derived simple sequence repeat (SSR) loci were validated for specific amplification and polymorphism across M. acuminata accessions. Fourteen loci were polymorphic, with alleles per polymorphic locus ranging from 3 to 8 and polymorphism information content ranging from 0.34 to 0.82.
CONCLUSIONS: A large set of unigenes were characterized in this study for both M. acuminata Calcutta 4 and Cavendish Grande Naine, increasing the number of public domain Musa ESTs. This transcriptome is an invaluable resource for furthering our understanding of biological processes elicited during biotic stresses in Musa. Gene-based markers will facilitate molecular breeding strategies, forming the basis of genetic linkage mapping and analysis of quantitative trait loci.

Matched MeSH terms: Ascomycota/genetics; Plant Diseases/genetics; Plant Leaves/genetics*; Musa/genetics*
Fulltext Expression patterns of genes involved in the defense and stress response of Spiroplasma citri infected Madagascar Periwinkle Catharanthus roseus

Nejat N, Vadamalai G, Dickinson M

Int J Mol Sci, 2012;13(2):2301-2313.
PMID: 22408455 DOI: 10.3390/ijms13022301

Madagascar periwinkle is an ornamental and a medicinal plant, and is also an indicator plant that is highly susceptible to phytoplasma and spiroplasma infections from different crops. Periwinkle lethal yellows, caused by Spiroplasma citri, is one of the most devastating diseases of periwinkle. The response of plants to S. citri infection is very little known at the transcriptome level. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate the expression levels of four selected genes involved in defense and stress responses in naturally and experimentally Spiroplasma citri infected periwinkles. Strictosidine β-glucosidase involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway showed significant upregulation in experimentally and naturally infected periwinkles. The transcript level of extensin increased in leaves of periwinkles experimentally infected by S. citri in comparison to healthy ones. A similar level of heat shock protein 90 and metallothionein expression was observed in healthy, naturally and experimentally spiroplasma-diseased periwinkles. Overexpression of Strictosidine β-glucosidase demonstrates the potential utility of this gene as a host biomarker to increase the fidelity of S. citri detection and can also be used in breeding programs to develop stable disease-resistance varieties.

Matched MeSH terms: Stress, Physiological/genetics*; Plant Leaves/genetics; Catharanthus/genetics*; Disease Resistance/genetics*

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