Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.
Blotting, Northern ; Genome ; Genome Size* ; Plant Viruses ; Product Packaging* ; RNA ; RNA, Viral ; Tobacco ; Tymovirus*

The dynamic activity of transposable elements (TEs) contributes to the vast diversity of genome size and architecture among plants. Here, we examined the genomic distribution and transposition activity of long terminal repeat retrotransposons (LTR-RTs) in Arabidopsis thaliana (Ath) and three of its relatives, Arabidopsis lyrata (Aly), Eutrema salsugineum (Esa), and Schrenkiella parvula (Spa), in Brassicaceae. Our analyses revealed the distinct evolutionary dynamics of Gypsyretrotransposons, which reflects the different patterns of genome size changes of the four species over the past million years. The rate of Gypsy transposition in Aly is approximately five times more rapid than that of Ath and Esa, suggesting an expanding Aly genome. Gypsy insertions in Esa are strictly confined to pericentromeric heterochromatin and associated with dramatic centromere expansion. In contrast, Gypsy insertions in Spa have been largely suppressed over the last million years, likely as a result of a combination of an inherent molecular mechanism of preferential DNA removal and purifying selection at Gypsy elements. Additionally, species-specific clades of Gypsy elements shaped the distinct genome architectures of Aly and Esa.
Brassicaceae/genetics* ; Evolution, Molecular ; Genome Size ; Genome, Plant ; Genomics ; Phylogeny ; Retroelements ; Species Specificity

Single-cell prokaryotes represent a simple and primitive cellular life form. The identification of the essential genes of bacteria and the minimal genome for the free-living cellular life could provide insights into the origin, evolution, and essence of life forms. The principles, methodology, and recent progresses in the identification of essential genes and minimal genome and the creation of synthetic cells are reviewed and particularly the strategies for creating the minimal genome and the potential applications are introduced.
Artificial Cells ; metabolism ; Bacteria ; genetics ; Genes, Essential ; Genome Size ; Genome, Bacterial ; genetics ; Metabolic Networks and Pathways ; genetics ; Synthetic Biology ; methods

Although the number of protein-coding genes is not highly variable between plant taxa, the DNA content in their genomes is highly variable, by as much as 2,056-fold from a 1C amount of 0.0648 pg to 132.5 pg. The mean 1C-value in plants is 2.4 pg, and genome size expansion/contraction is lineage-specific in plant taxonomy. Transposable element fractions in plant genomes are also variable, as low as ~3% in small genomes and as high as ~85% in large genomes, indicating that genome size is a linear function of transposable element content. Of the 2 classes of transposable elements, the dynamics of class 1 long terminal repeat (LTR) retrotransposons is a major contributor to the 1C value differences among plants. The activity of LTR retrotransposons is under the control of epigenetic suppressing mechanisms. Also, genome-purging mechanisms have been adopted to counter-balance the genome size amplification. With a wealth of information on whole-genome sequences in plant genomes, it was revealed that several genome-purging mechanisms have been employed, depending on plant taxa. Two genera, Lilium and Fritillaria, are known to have large genomes in angiosperms. There were twice times of concerted genome size evolutions in the family Liliaceae during the divergence of the current genera in Liliaceae. In addition to the LTR retrotransposons, non-LTR retrotransposons and satellite DNAs contributed to the huge genomes in the two genera by possible failure of genome counter-balancing mechanisms.
Angiosperms ; Classification ; DNA ; DNA Transposable Elements* ; DNA, Satellite ; Epigenomics ; Fritillaria ; Genome ; Genome Size* ; Genome, Plant ; Humans ; Liliaceae ; Lilium ; Plants ; Retroelements ; Terminal Repeat Sequences

The method of cell lysate preparation of M. leprae is an important technique in the study of leprosy. This report describes the optimization of method for cell lysate preparation of M. leprae obtained from infected nude mouse. After M. leprae isolated from nude mouse foot-pad was disrupted by sonication, it was centrifuged and then whole lysate was prepared. With this method it was possible to isolate 0.3 mg whole cell lysate using 20 mg of M. leprae. By SDS-PAGE and Coomassie blue staining, the number of protein in M. leprae is less than that of other bacteria, for example, E. coli and M. smegmatis. It is likely that this is due to the small genome size. This work will contribute to the analysis of new protein antigen of M. leprae and the basic study for the development of vaccine in leprosy.
Animals ; Bacteria ; Electrophoresis, Polyacrylamide Gel ; Genome Size ; Leprosy ; Mice ; Mice, Nude* ; Sonication

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organ systems. Although the etiology of SLE remains unclear, it is widely accepted that genetic factors could be involved in its pathogenesis. A number of genome-wide association studies (GWASs) have identified novel single-nucleotide polymorphisms (SNPs) associated with the risk of SLE in diverse populations. However, not all the SNP candidates identified from non-Asian populations have been validated in Koreans. In this study, we aimed to replicate the SNPs that were recently discovered in the GWAS; these SNPs have not been validated in Koreans or have only been replicated in Koreans with an insufficient sample size to conclude any association. For this, we selected five SNPs (rs1801274 in FCGR2A and rs2286672 in PLD2, rs887369 in CXorf21, rs9782955 in LYST, and rs3794060 in NADSYN1). Through the replication study with 656 cases and 622 controls, rs1801274 in FCGR2A was found to be significantly associated with SLE in Koreans (odds ratio, 1.26, 95% confidence interval, 1.06 to 1.50; p = 0.01 in allelic model). This association was also significant in two other models (dominant and recessive). The other four SNPs did not show a significant association. Our data support that FCGR polymorphisms play important roles in the susceptibility to SLE in diverse populations, including Koreans.
Autoimmune Diseases ; Genome-Wide Association Study ; Lupus Erythematosus, Systemic ; Polymorphism, Single Nucleotide ; Sample Size

Power and sample size estimation is one of the crucially important steps in planning a genetic association study to achieve the ultimate goal, identifying candidate genes for disease susceptibility, by designing the study in such a way as to maximize the success possibility and minimize the cost. Here we review the optimal two-stage genotyping designs for genomewide association studies recently investigated by Wang et al(2006). We review two mathematical frameworks most commonly used to compute power in genetic association studies prior to the main study: Monte-Carlo and non-central chi-square estimates. Statistical powers are computed by these two approaches for case-control genotypic tests under one-stage direct association study design. Then we discuss how the linkagedisequilibrium strength affects power and sample size, and how to use empirically-derived distributions of important parameters for power calculations. We provide useful information on publicly available softwares developed to compute power and sample size for various study designs.
Sample Size ; Research Design ; *Models, Statistical ; Humans ; *Genome, Human ; Genetic Screening

Helicobacter pylori is a causative agent of type B gastritis and plays a central role in the pathogenesis of gastroduodenal ulcers and gastric cancer. Strategies for the control of H. pylori- induced gastroduodenal diseases based on conventional measures are still of limited utility. Therefore, it seems worthwhile to make a break-through as an alternative strategy by reviewing the host-parasite relationship of H. pylori infection on the basis of genomic structure. In this study, we tried to construct a physical map of H. pylori genome. Chromosomal DNA from a Korean prototype strain, H. pylori 51 was digested with 42 restriction endonucleases to identify restriction patterns suitable for mapping the genome. We identified three enzymes, ApaI, NotI and Sfil, which gave a small number of DNA fragments of higher molecular weight that were well resolved after pulsed-field gel electrophoresis. The H. pylori chromosome contained 7 ApaI fragments ranging from 167 to 311 kb, 7 NotI fragments ranging from 5 to 516 kb and 2 SfiI fragments of 332 and 1,347 kb in size. The genome size of the strain is 1,679 kb. A circular physical map of the H. pylori chromosome was constructed by aligning 3 kinds of restriction fragments by Southern blot analysis of simple ApaI, NotI and SfiI digests or double NotI/ApaI and NotI/SfiI digests with the various probes. When the physical map of H. pylori strain 51 compared with that of strain 26695 of which the cornplete genome sequence was reported, completely different restriction patterns were shown, which suggests the genomic diversity in H. pylori.
Blotting, Southern ; DNA ; DNA Restriction Enzymes ; Electrophoresis, Gel, Pulsed-Field ; Gastritis ; Genome ; Genome Size ; Helicobacter pylori* ; Helicobacter* ; Host-Parasite Interactions ; Molecular Weight ; Peptic Ulcer ; Stomach Neoplasms

Zika virus (ZIKV) is one of the pathogens which is transmitted world widely, but there are no effective drugs and vaccines. Whole genome sequencing (WGS) of viruses could be applied to viral pathogen characterization, diagnosis, molecular surveillance, and even finding novel pathogens. We established an improved method using direct RNA sequencing with Nanopore technology to obtain WGS of ZIKV, after adding poly (A) tails to viral RNA. This established method does not require specific primers, complimentary DNA (cDNA) synthesis, and polymerase chain reaction (PCR)-based enrichment, resulting in the reduction of biases as well as of the ability to find novel RNA viruses. Nanopore technology also allows to read long sequences. It makes WGS easier and faster with long-read assembly. In this study, we obtained WGS of two strains of ZIKV following the established protocol. The sequenced reads resulted in 99% and 100% genome coverage with 63.5X and 21,136X, for the ZIKV PRVABC59 and MR 766 strains, respectively. The sequence identities of the ZIKV PRVABC59 and MR 766 strains for each reference genomes were 98.76% and 99.72%, respectively. We also found that the maximum length of reads was 10,311 bp which is almost the whole genome size of ZIKV. These long-reads could make overall structure of whole genome easily, and WGS faster and easier. The protocol in this study could provide rapid and efficient WGS that could be applied to study the biology of RNA viruses including identification, characterization, and global surveillance.
Bias (Epidemiology) ; Biology ; Diagnosis ; DNA ; Genome ; Genome Size ; Methods ; Nanopores ; Polymerase Chain Reaction ; RNA Viruses ; RNA ; RNA, Viral ; Sequence Analysis, RNA ; Tail ; Vaccines ; Zika Virus

Diabetes is a common metabolic disorder with a worldwide prevalence of 8.3% and is the leading cause of visual loss, end-stage renal disease and amputation. Recently, genome-wide association studies (GWASs) have identified genetic risk factors for diabetic microvascular complications of retinopathy, nephropathy, and neuropathy. We summarized the recent findings of GWASs on diabetic microvascular complications and highlighted the challenges and our opinion on future directives. Five GWASs were conducted on diabetic retinopathy, nine on nephropathy, and one on neuropathic pain. The majority of recent GWASs were underpowered and heterogeneous in terms of study design, inclusion criteria and phenotype definition. Therefore, few reached the genome-wide significance threshold and the findings were inconsistent across the studies. Recent GWASs provided novel information on genetic risk factors and the possible pathophysiology of diabetic microvascular complications. However, further collaborative efforts to standardize phenotype definition and increase sample size are necessary for successful genetic studies on diabetic microvascular complications.
Amputation ; Diabetic Retinopathy ; Genetics ; Genome-Wide Association Study* ; Kidney Failure, Chronic ; Neuralgia ; Phenotype ; Prevalence ; Risk Factors ; Sample Size