First report on prevalence, molecular characterization and phylogenetic study of Toxoplasma gondii infecting sheep of the Malakand Division of Pakistan.

BACKGROUND: Toxoplasma gondii is an apicomplexan protozoan parasite that infects one-third of the population of the world, including humans, animals, birds, and other vertebrates. The present investigation is the first molecular attempt in the Malakand Division of Pakistan to determine the epidemiology and phylogenetic study of Toxoplasma gondii infecting small ruminants. METHODOLOGY: A total of (N = 450) blood samples of sheep were randomly collected during the study period (December 2020 to November 2021), and DNA detection was done using PCR by amplifying ITS-1 genes. SPSS.20 and MEGA-11 software were used for statistical significance and phylogenetic analysis. RESULTS: The overall prevalence of T. gondii infection among sheep was 14.44 % (65/450). A high infection rate was found in more than five-year-olds at 18.33 % (11/60). Sequencing and BLAST analysis of PCR-positive samples confirmed the presence of T. gondii. Randomly, three isolates were sequenced and submitted to GenBank under accession numbers (PP028089-PP028091), respectively. The BLAST analysis of the obtained sequences based on the ITS-1 gene showed 99 % similarities with reported genotypes found in goats of Malakand, Pakistan (PP028089) and dogs of Brazil (MF766454). The study concludes that T. gondii is notably prevalent among the sheep population in the region, emphasizing the significant role of risk factors in disease transmission across animals and potentially to humans. Further research, zoonotic potential analysis, and targeted control measures are warranted to address and manage this parasitic infection effectively.

An mRNA-based reverse-vaccinology strategy to stimulate the immune response against Nipah virus in humans using fusion glycoproteins.

The zoonotic pathogen, Nipah virus, is considered a potential healthcare threat due to its high mortality rates and detrimental symptoms like encephalitis. Ribavirin, an antiviral drug helps in overcoming the number of casualties and reducing the mortality rate, but no long-lasting solution has been proposed yet putting global health security in jeopardy. Given the cognizance of mRNA-based vaccines as safe and efficacious preventative strategies against pathogens, the current study has utilized the reverse-vaccinology approach coupled with immunoinformatics to propose an mRNA-based vaccine candidate against the Nipah virus. To ensure the effectiveness of the vaccine candidate against all strains of Nipah and associated viruses, three fusion glycoproteins from Nipah and Hendra viruses were selected. A total of 30 potential epitopes, 10 B-cell-, 10 MHC-I-, and 10 MHC-II-specific, were screened for the construct. The finalized epitopes were highly antigenic with scores ranging from 0.75 to 1.7615 at a threshold of 0.4 for viruses and non-homologous to Homo sapiens eradicating any chance of immune tolerance. The construct, with a World population coverage of 97.2%, was structurally stable, thermostable, and hydrophilic with indices of 32.91, 93.62, and -0.002, respectively. The vaccine candidate's tertiary structure was predicted with a TM score of 0.131 and the refined model displayed superlative RAMA improvement (98.2) and MolProbity score (0.975). A quality factor of 93.5421% further validated the structural quality and stability. A prompt and stable immune response was also simulated, and the vaccine candidate was shown to eliminate from the body within the first five days of injection. Immune complexes count of 7000 mg/mL was predicted against the antigen with a small but nonsignificant danger signal, countered by the cytokines. Lastly, strong molecular interactions of the vaccine candidate with TLR-3 (331.09 kcal/mol) and TLR-4 (-333.31 kcal/mol) and molecular dynamics simulation analysis authenticated the immunogenic potential of the vaccine candidate. This vaccine candidate can serve as a foundation for future in-vitro and in-vivo trials to minimize or eradicate the diseases associated with the Nipah virus or the Henipaviral family.

An Insight into the Role of E2F1 in Breast Cancer Progression, Drug Resistance, and Metastasis.

AIMS: This study aimed to investigate the role of E2F1 in breast cancer biology. BACKGROUND: Expression of E2F1, a transcription factor of many oncogenes and tumor suppressor genes, is lowered in several malignancies, including breast carcinoma. OBJECTIVES: In the present study, we analyzed the status of E2F1 expression in association with diverse attributes of breast malignancy and its impact on cancer progression. METHODS: For this purpose, we used various freely available online applications for gene enrichment, expression, and methylation analysis to extract mutation-based E2F1 map, to measure E2F1 drug sensitivity, and to determine E2F1 association with DNA damage response proteins. RESULTS: Results revealed tissue-specific regulatory behavior of E2F1. Moreover, the key role of E2F1 in the promotion of metastasis, stem cell-mediated carcinogenesis, estrogen-mediated cell proliferation, and cellular defense system, has therefore highlighted it as a metaplastic marker and hot member of key resistome pathways. CONCLUSION: The information thus generated can be employed for future implications in devising rational therapeutic strategies. Moreover, this study has provided a more detailed insight into the diagnostic and prognostic potential of E2F1.

Unraveling the possible inhibitors for Chorismate synthase to combat tuberculosis using in silico approach.

Tuberculosis antibiotic resistance is a huge concern to the global population. The goal of this study was to find new and effective compounds to treat multidrug-resistant tuberculosis by targeting Chorismate synthase (CS), a crucial enzyme for Mycobacterium tuberculosis survival (MbT). The potential of a library of compounds as selective anti - tuberculosis drugs was investigated. Docking was first conducted using MoE to determine the effectiveness of the compounds. Molecular docking studies followed by MD simulation studies (total of 500 ns) in combination with free energy calculations grade the ligands in terms of their binding affinities. In the ligand bound state of the CS, MD simulations revealed a change from stretched to bended motional shift in loop L19. The RMSF analysis also revealed this flexibility, which was confirmed by visual inspection of L19 at various time intervals during the experiment. It appears that ZF1(-25.43Kcal/mol) and ZF2 (-22.04Kcal/mol) form hbonds and have a high binding energy in the active region of protein. Residues wise distribution of binding energy reveals that Arg144, Trp4, Thr6, and L19 amino acid residues are engaged in binding of CS with inhibitors. In summary, the findings suggest that compounds ZF1 and ZF2 may be more effective and selective anti-TB agents than currently available drugs. Also the role of L19, mediated by αH9 and αH5 in the retention of ligand inside the active pocket, through the formation of lid was also revealed. This knowledge will aid in the discovery of drugs that are potent CS inhibitors. More experimental research and a better understanding of the structure-activity relationship could aid in the development of possible candidates with better CS inhibition.Communicated by Ramaswamy H. Sarma.

Exceptional behavior of breast cancer-associated type 1 gene in breast invasive carcinoma.

Background: Cellular expression level of Breast Cancer-Associated Type 1 (BRCA1) encoded protein is the sign of genome integrity, stability, and surveillance. BRCA1 after sensing DNA damage activates repairing system and if mutated leaves genomic lesions unrepaired and triggers transformation of normal breast cells into cancerous ones. Aims of study: We conducted in silico study to have a holistic view of BRCA1's correlation with multiple variables of breast invasive carcinoma. Materials and Methods: We used user-friendly online GeneCardsSuite pathway-level enrichment analysis, UALCAN portal differential expression analysis, cBioPortal cancer genome platform for mutatome map construction, and cancer cell lines encyclopedia genomics of drug sensitivity toolkit to understand correlation of BRCA1 expression with the effectiveness of anti-cancer drugs. Results: Contrary to general behavior of a tumor suppressor gene our study revealed BRCA1 overexpression under all circumstances. This novel finding needs to be explored further to understand functional impact of BRCA1 overexpression on the expression of many genes which are transcriptionally regulated by BRCA1 and promotion of tumriogenesis. Conclusion: Our study highlights the potential role of BRCA1-regulated genes in oncogenesis and recommends use of BRCA1-linked genes as future therapeutic targets for effective disease management.

Exploring multi-target inhibitors using in silico approach targeting cell cycle dysregulator-CDK proteins.

Cyclin-dependent kinases (CDKs) belong to a family of multifunctional enzymes that control cell cycle modifications, transcription, and cell proliferation. Their dysfunctions result in different diseases like cancer making them an important drug target in oncology and beyond. The present study aims at identifying the selective inhibitors for ATP binding site in CDK proteins (CDK1, CDK2, CDK4, and CDK5) following a multi-target drug designing approach. Significant challenges lie in identifying the selective inhibitor for the ATP binding site as this region is highly conserved in all protein kinases. Molecular docking coupled with molecular dynamics simulation and free energy of binding calculations (MMPBSA/MMGBSA) were used to identify the potent competitive ATP binding site inhibitors. All the four proteins were docked against the library of drug-like compounds and the outcomes of the docking study were further analyzed by Molecular dynamics (total of 6µs) and MMPB/GBSA techniques. Five different inhibitors for structurally distant protein kinases, i.e. CDK1, CDK2, CDK4, and CDK5 are identified with the binding energy (ΔGbind-PB) in the range -18.24 to -28.43Kcal/mol. Mechanistic complexities associated with the binding of the inhibitor are unraveled by carefully analyzing the MD trajectories. It is observed that certain residues (Lys33, Asp127, Asp145, Tyr15, Gly16, Asn144) and regions are critical for the retention of inhibitors in active pocket, and significant conformational changes take place in the active site region as well as its neighbor following the entry of the ligand inside active pocket as inferred by RMSD and RMSF. It is observed that LIG3 and LIG4 are the best possible inhibitors as reflected from their high binding energy, interaction pattern, and their retention inside the active pocket. This study will facilitate the process of multi-target drug designing against CDK proteins and can be used in the development of potential therapeutics against different diseases.

Targeting protein tyrosine phosphatase to unravel possible inhibitors for Streptococcus pneumoniae using molecular docking, molecular dynamics simulations coupled with free energy calculations.

AIMS: Protein tyrosine phosphatase (PTP-CPS4B) is a signaling enzyme that is essential for a wide range of cellular processes, like metabolism, proliferation, survival and motility. Studies suggest that PTPs are vital for the production of Wzy-dependent capsule in bacteria, making it a valuable target for the discovery of pneumonia associated anti-virulence antibacterial agents. Present study aims at identifying the potential drug candidates to be exploited in inhibiting the growth of Streptococcus pneumonia targeting PTP-CPS4B. MATERIALS AND METHODS: The present study exploits the molecular docking potential coupled with molecular dynamic simulation as well as free energy calculations to identify potential inhibitors of PTP-CPS4B. Libraries of known and unknown compounds were docked into the active site of PTP-CPS4B using MOE. The compounds with best binding affinity and orientation were subjected to MD simulations and free energy calculations. FINDINGS: Top three compounds based on their binding energy and well composed interaction pattern obtained from molecular docking study were subjected to MD simulations and were compared to reported antibiotic drugs. MD Simulation studies have shown that the presence of an inhibitor inside the active site reduces protein flexibility as evident from RMSD, RMSF and Principal component analyses. MD simulations identified a transition from extended to bended motional shift in loop α6 of the PTP-CPS4B in ligand bound state. This flexibility was reported in the RMSF analysis and verified by the visual investigation of the loop α6 at different time intervals during the simulation. Free energy of binding affinity (computed using MMPBSA &MMGBSA approach) and the interaction patterns obtained from MD trajectory indicate that compound ZN1 (-31.50 Kcal/mol), ZN2 (-33.14 Kcal/mol) and ZN3 (-26.60 Kcal/mol) are potential drug candidates against PTP-CPS4B. Residue wise decomposition study helped in identifying the role of individual amino acid towards the overall inhibition behavior of the compounds. PCA analysis has led to the conclusion that the behavior of PTP-CPS4B inhibitors causes conformational dynamics that can be used to describe the protein inhibition mechanism. SIGNIFICANCE: The outcome reveals that this study provide enough evidences for the consideration of ZN1, ZN2, ZN3 as potential PTP-CPS4B inhibitors and further in vitro and in vivo studies may prove their therapeutic potential.

nCOV-19 peptides mass fingerprinting identification, binding, and blocking of inhibitors flavonoids and anthraquinone of Moringa oleifera and hydroxychloroquine.

An rare pandemic of viral pneumonia occurs in December 2019 in Wuhan, China, which is now recognized internationally as Corona Virus Disease 2019 (COVID-19), the etiological agent classified as Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). According to the World Health Organization (WHO), it has so far expanded to more than 213 countries/territories worldwide. Our study aims to find the viral peptides of SARS-COV-2 by peptide mass fingerprinting (PMF) in order to predict its novel structure and find an inhibitor for each viral peptide. For this reason, we calculated the mass of amino acid sequences translated from the SARS-CoV2 whole genome and identify the peptides that may be a target for inhibition. Molecular peptide docking with Moringa oleifera, phytochemicals (aqueous and ethanolic) leaf extracts of flavonoids (3.56 ± 0.03), (3.83 ± 0.02), anthraquinone (11.68 ± 0.04), (10.86 ± 0.06) and hydroxychloroquine present therapy of COVID-19 in Pakistan for comparative study. Results indicate that 15 peptides of SARS-CoV2 have been identified from PMF, which is then used as a selective inhibitor. The maximum energy obtained from AutoDock Vina for hydroxychloroquine is -5.1 kcal/mol, kaempferol (flavonoid) is -6.2 kcal/mol, and for anthraquinone -6 kcal/mol. Visualization of docking complex, important effects are observed regarding the binding of peptides to drug compounds. In conclusion, it is proposed that these compounds are effective antiviral agents against COVID-19 and can be used in clinical trials.Communicated by Ramaswamy H. Sarma.

Dynamics and conformational propensities of staphylococcal CntA.

Enzymes use transition metals as co-factors for catalytic roles in biological processes. Notably, manganese, iron, cobalt, nickel, copper and zinc are abundantly used. Staphylococcus aureus, a commensal bacterium asymptomatically, lies on the human body causing variety of infections. S. aureus is equipped by advanced virulence-regulatory circuits of metal acquisition like Cnt that acquires metals at infection sites by utilizing a nicotianamine-like metallophore staphylopine. Despite significant growth in structural studies, how CntA of Cnt system transmits conformational signal upon staphylopine recognition remains elusive. Here, we analyzed the structural changes adopted by CntA during close-to-open transition by computational approaches. CntA uses a bi-domain architectural form of domain II which performed 37° rigid body rotation and 1.1 Å translation assisted by inter-domain hinge cluster residues. Important clustered communities were found regulating the conformational changes in CntA where communities 4 and 5 are found crucial. Besides open and close states, the fluctuating regions sampled two additional intermediate states which were considered close or open previously. CntA prefers fluctuating the non-conserved regions rather than conserved where domain II turned out to be rigid and maintains a stable fold. Overall, the CntA system is a potential target for structural biologist to hamper such conformational behaviors at family level.Communicated by Ramaswamy H. Sarma.

The complete mitochondrial genome of blue pansy, Junonia orithya (Lepidoptera: Nymphalidae: Nymphalinae) from Pakistan.

Junonia orithya's complete mitochondrial genome (mitogenome) is determined to be 14,214 bp in length, including 37 typical mitochondrial genes and an AT-rich region. Its gene order and orientation are identical to those of other butterfly species. All PCGs are initiated by typical ATN codons, except for cox1 gene which is started by CGA codon. Nine genes use complete termination codon (TAA), whereas the COX1, COX2, NADH1 and NAH4 genes end with single T. Except for trnS1(AGN), all tRNA genes display typical secondary cloverleaf structures as those of other insects. The 331 bp long AT-rich region contains several features common to the other lepidopterans, such as the ATAGA motif followed by a 18 bp poly-T stretch, two microsatellite-like (TA) 9 elements, a 5 bp poly-A stretch immediately upstream of trNAM gene from Pakistan.

Phylogenetic association of Cyprinid (Schizothorax) inferred from complete mitochondrial genome.

Whole mitochondrial DNA (mtDNA) of S.esocinusand S. plagiostomus was found to be 16,591 and 16,564 bp respectively with 13 protein-coding genes, 2 rRNAs genes, 22 tRNA genes and 2 non-coding region. For valuing the phylogenetic relationship of a species on the basis of whole mitogenome are considered to be a great importance. In this study, we sequenced whole mitogenome of S.esocinusand S. plagiostomus and compared with the whole mitogenome sequences of members of other families (Balitoridae, Nemanchillidae and Cobatidae). The monophyly of the family Cyprinidae and a clade comprising Balitoridae and Nemanchillidae while Cobatidae with paraphyletic origin was strongly supported by the resultant phylogenies, recognized that Cyprinidae was closely related with the family Cobatidae than other ones. The results indicated that whole mitochondrial genome has a great importance in studying variation in genes and phylogenetic relationship in the subfamily Schizothoracinae. This data offering the molecular phylogenetic frame work of important Schizothorax species found in Swat.

Comparative phylogenetic analysis of Schizothorax plagiostomus and Schizothorax esocinus with other members of subfamilies of cyprinidae on the basis of complete mitochondrial genome and 12S, 16S ribosomal RNA from Northren areas of Pakistan.

We assessed the relationship of Schizothoracinae species with other subfamilies Alburninae, Xenocyprinae, Cultrinae and Squaliobarbinae of family Cyprinidae by creating the phylogenetic trees using complete mitogenome and 12S and 16S RNA. Our representative species show the great affiliation with other but separated from a group composed of Metzia mesembrinum, Metzia longinasus, Metzia lineata and Metzia formosae of subfamily Alburninae while other subfamilies formed distinct group. The members of subfamily Schizothoracinae shows separate line of evolution from subfamily Barbinae.

A review on Zika virus outbreak, epidemiology, transmission and infection dynamics.

Zika virus (ZIKV) is a newly emergent relative of the Flaviviridae family and linked to dengue (DENV) and Chikungunya (CHIVKV). ZIKV is one of the rising pathogens promptly surpassing geographical borders. ZIKV infection was characterized by mild disease with fever, headache, rash, arthralgia and conjunctivitis, with exceptional reports of an association with Guillain-Barre syndrome (GBS) and microcephaly. However, since the end of 2015, an increase in the number of GBS associated cases and an astonishing number of microcephaly in fetus and new-borns in Brazil have been related to ZIKV infection, raising serious worldwide public health concerns. ZIKV is transmitted by the bite of infected female mosquitoes of Aedes species. Clarifying such worrisome relationships is, thus, a current unavoidable goal. Here, we extensively described the current understanding of the effects of ZIKV on heath, clinical manifestation, diagnosis and treatment options based on modern, alternative and complementary medicines regarding the disease.

Comparative phylogeny of (Schizothorax esocinus) with reference to 12s and 16 sribosomal RNA from River Swat, Pakistan.

Schizothorax esocinus is one of the important cold water delicious fish belonging to family Cyprinidae and found in River Swat which is one of the main river flowing in Khyber Pakhtunkhwa, Pakistan.The complete mitochondrial genomes of the S. esocinus species was found to be 16,591bp. Phylogenetic analysis of S. esocinus based on 12 s ribosomal RNA and 16S rRNA confirmed that, the phylogenetic position of S. esocinus was slightly different, but clustered closed to S. plagiostomus, S. labiatus, S. richardsonii, S. nepalensis, and S. progastus predicting a close homology in genome and hence a monophyletic line of evolution also this specie showed close relationship and homologs to the Aspiorhynchus laticeps, Spinibarbus sinensis, Aspiorhynchus laticeps, and Percocypris respectively. This study provided phylogenetic relationship between Schizothoracinae fishes and with the other fishes of family cyprinidae even at the subfamily level.

Two-component systems regulate ABC transporters in antimicrobial peptide production, immunity and resistance.

Bacteria offer resistance to a broad range of antibiotics by activating their export channels of ATP-binding cassette transporters. These transporters perform a central role in vital processes of self-immunity, antibiotic transport and resistance. The majority of ATP-binding cassette transporters are capable of detecting the presence of antibiotics in an external vicinity and are tightly regulated by two-component systems. The presence of an extracellular loop and an adjacent location of both the transporter and two-component system offers serious assistance to induce a quick and specific response against antibiotics. Both systems have demonstrated their ability of sensing such agents, however, the exact mechanism is not yet fully established. This review highlighted the three key functions of antibiotic resistance, transport and self-immunity of ATP-binding cassette transporters and an adjacent two-component regulatory system.

Interspecific studies of circadian genes period and timeless in Drosophila.

The level of rescue of clock function in genetically arrhythmic Drosophila melanogaster hosts using interspecific clock gene transformation was used to study the putative intermolecular coevolution between interacting clock proteins. Among them PER and TIM are the two important negative regulators of the circadian clock feedback loop. We transformed either the D. pseudoobscura per or tim transgenes into the corresponding arrhythmic D. melanogaster mutant (per01 or tim01) and observed >50% rhythmicity but the period of activity rhythm was either longer (D. pseudoobscura-per) or shorter than 24 h (D. pseudoobscura-tim) compared to controls. By introducing both transgenes simultaneously into double mutants, we observed that the period of the activity rhythm was rescued by the pair of hemizygous transgenes (~24 h). These flies also showed a more optimal level of temperature compensation for the period. Under LD 12:12 these flies have a D. pseudoobscura like activity profile with the absence of morning anticipation as well as a very prominent earlier evening peak of activity rhythm. These observation are consistent with the view that TIM and PER form a heterospecific coevolved module at least for the circadian period of activity rhythms. However the strength of rhythmicity was reduced by having both transgenes present, so while evidence for a coevolution between PER and TIM is observed for some characters it is not for others.

Identification and characterization of mobile genetic elements LINEs from Brassica genome.

Among transposable elements (TEs), the LTR retrotransposons are abundant followed by non-LTR retrotransposons in plant genomes, the lateral being represented by LINEs and SINEs. Computational and molecular approaches were used for the characterization of Brassica LINEs, their diversity and phylogenetic relationships. Four autonomous and four non-autonomous LINE families were identified and characterized from Brassica. Most of the autonomous LINEs displayed two open reading frames, ORF1 and ORF2, where ORF1 is a gag protein domain, while ORF2 encodes endonuclease (EN) and a reverse transcriptase (RT). Three of four families encoded an additional RNase H (RH) domain in pol gene common to 'R' and 'I' type of LINEs. The PCR analyses based on LINEs RT fragments indicate their high diversity and widespread occurrence in tested 40 Brassica cultivars. Database searches revealed the homology in LINE sequences in closely related genera Arabidopsis indicating their origin from common ancestors predating their separation. The alignment of 58 LINEs RT sequences from Brassica, Arabidopsis and other plants depicted 4 conserved domains (domain II-V) showing similarity to previously detected domains. Based on RT alignment of Brassica and 3 known LINEs from monocots, Brassicaceae LINEs clustered in separate clade, further resolving 4 Brassica-Arabidopsis specific families in 2 sub-clades. High similarities were observed in RT sequences in the members of same family, while low homology was detected in members across the families. The investigation led to the characterization of Brassica specific LINE families and their diversity across Brassica species and their cultivars.

The landscape and structural diversity of LTR retrotransposons in Musa genome.

Long terminal repeat retrotransposons represent a major component of plant genomes and act as drivers of genome evolution and diversity. Musa is an important fruit crop and also used as a starchy vegetable in many countries. BAC sequence analysis by dot plot was employed to investigate the LTR retrotransposons from Musa genomes. Fifty intact LTR retrotransposons from selected Musa BACs were identified by dot plot analysis and further BLASTN searches retrieved 153 intact copies, 61 truncated, and a great number of partial copies/remnants from GenBank database. LARD-like elements were also identified with several copies dispersed among the Musa genotypes. The predominant elements were the LTR retrotransposons Copia and Gypsy, while Caulimoviridae (pararetrovirus) were rare in the Musa genome. PCR amplification of reverse transcriptase (RT) sequences revealed their abundance in almost all tested Musa accessions and their ancient nature before the divergence of Musa species. The phylogenetic analysis based on RT sequences of Musa and other retrotransposons clustered them into Gypsy, Caulimoviridae, and Copia lineages. Most of the Musa-related elements clustered in their respective groups, while some grouped with other elements indicating homologous sequences. The present work will be helpful to understand the LTR retrotransposons landscape, giving a complete picture of the nature of the elements, their structural features, annotation, and evolutionary dynamics in the Musa genome.

Evolutionary genomics of miniature inverted-repeat transposable elements (MITEs) in Brassica.

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in most eukaryotic genomes. We aimed to characterize various MITEs families in Brassica in terms of their presence, sequence characteristics and evolutionary activity. Dot plot analyses involving comparison of homoeologous bacterial artificial chromosome (BAC) sequences allowed identification of 15 novel families of mobile MITEs. Of which, 5 were Stowaway-like with TA Target Site Duplications (TSDs), 4 Tourist-like with TAA/TTA TSDs, 5 Mutator-like with 9-10 bp TSDs and 1 novel MITE (BoXMITE1) flanked by 3 bp TSDs. Our data suggested that there are about 30,000 MITE-related sequences in Brassica rapa and B. oleracea genomes. In situ hybridization showed one abundant family was dispersed in the A-genome, while another was located near 45S rDNA sites. PCR analysis using primers flanking sequences of MITE elements detected MITE insertion polymorphisms between and within the three Brassica (AA, BB, CC) genomes, with many insertions being specific to single genomes and others showing evidence of more recent evolutionary insertions. Our BAC sequence comparison strategy enables identification of evolutionarily active MITEs with no prior knowledge of MITE sequences. The details of MITE families reported in Brassica enable their identification, characterization and annotation. Insertion polymorphisms of MITEs and their transposition activity indicated important mechanism of genome evolution and diversification. MITE families derived from known Mariner, Harbinger and Mutator DNA transposons were discovered, as well as some novel structures. The identification of Brassica MITEs will have broad applications in Brassica genomics, breeding, hybridization and phylogeny through their use as DNA markers.