Structural variation of the malaria-associated human glycophorin A-B-E region

Approximately 5% of the human genome consists of structural variants, which are enriched for genes involved in the immune response and cell-cell interactions. A well-established region of extensive structural variation is the glycophorin gene cluster, comprising three tandemly-repeated regions about 120kb in length, carrying the highly homologous genes GYPA, GYPB and GYPE. Glycophorin A and glycophorin B are glycoproteins present at high levels on the surface of erythrocytes, and they have been suggested to act as decoy receptors for viral pathogens. They act as receptors for invasion of a causative agent of malaria, Plasmodium falciparum. A particular complex structural variant (DUP4) that creates a GYPB/GYPA fusion gene is known to confer resistance to malaria. Many other structural variants exist, and remain poorly characterised. Here, we analyse sequences from 6466 genomes from across the world for structural variation at the glycophorin locus, confirming 15 variants in the 1000 Genomes project cohort, discovering 9 new variants, and characterising a selection using fibre-FISH and breakpoint mapping. We identify variants predicted to create novel fusion genes and a common inversion duplication variant at appreciable frequencies in West Africans. We show that almost all variants can be explained by unequal cross over events (non-allelic homologous recombination, NAHR) and. by comparing the structural variant breakpoints with recombination hotspot maps, show the importance of a particular meiotic recombination hotspot on structural variant formation in this region.

Evolutionary and functional analysis of RBMY1 gene copy number variation on the human Y chromosome

Human RBMY1 genes are located in four variable-sized clusters on the Y chromosome, expressed in male germ cells and possibly associated with sperm motility. We have re-investigated the mutational background and evolutionary history of the RBMY1 copy number distribution in worldwide samples and its relevance to sperm parameters in an Estonian cohort of idiopathic male factor infertility subjects. We estimated approximate RBMY1 copy numbers in 1218 1000 Genomes Project phase 3 males from sequencing read-depth, then chose 14 for valid ation by multicolour fibre-FISH. These fibre-FISH samples provided accurate calibration standards for the entire panel and led to detailed insights into population variation and mutational mechanisms. RBMY1 copy number worldwide ranged from 3 to 13 with a mode of 8. The two larger proximal clusters were the most variable, and additional duplications, deletions and inversions were detected. Placing the copy number estimates onto the published Y-SNP-based phylogeny of the same samples suggested a minimum of 562 mutational changes, translating to a mutation rate of 2.20 × 10−3 (95% CI 1.94 × 10−3 to 2.48 × 10−3) per father-to-son Y-transmission, higher than many short tandem repeat (Y-STRs), and showed no evidence for selection for increased or decreased copy number, but possible copy number stabilizing selection. An analysis of RBMY1 copy numbers among 376 infertility subjects failed to replicate a previously reported association with sperm motility and showed no significant effect on sperm count and concentration, serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone levels or testicular and semen volume. These results provide the first in-depth insights into the structural rearrangements underlying RBMY1 copy number variation across diverse human lineages.

Epi-fluorescence Microscopy of Single Molecule DNA Denaturation in situ

DNA can be denatured by two main methods which are: a) denaturation in solution (invitro) and b) denaturation on a slide surface (in-situ). Additionally, DNA can also be denatured in gels with urea. The method to be used depends on various factors such as the application, the source of the DNA, the length, and the techniques available to confirm the extent of denaturation. Verification of the extent of denaturation is important because of the following factors: 1) increases the chances of hybridization (especially for short probes), 2) prevents the loss of expensive probes (if the target site is not denatured then, the probes will not hybridize and will only cause a high a background), 3) a higher degree of denaturation allows for more probes to be used and therefore, more information can be derived after hybridization, and 4) essential to maximize due to extremely short probe length. It is important to ensure that DNA morphology is preserved after denaturation in order for the probes to hybridise and also for ensuring proper statistical analysis for high throughput applications. In this work, various experimental conditions for in situ denaturation of single molecule DNA is presented.Significance StatementThe significance of this work is that it emphasizes on the importance of denaturation of target genomic DNA in DNA fibre FISH (fluorescence in situ hybridisation) experiments. If the quality of the target DNA is poor after denaturation or the target DNA is not properly denatured, then it will be very difficult or impossible to hybridize the probe DNA during FISH experiments. This will affect the final results for DNA FISH. Additionally, it is the first time that single DNA combed molecules have been shown to be denatured in situ. Most of the past work has been on gels only. Thus the work is both unique and significant.

High resolution molecular cytogenetic techniques in plants: Pachytene- and fibre-FISH

Fluorescence in situ hybridization (FISH) is the most versatile and accurate molecular cytogenetic technique for determining euchromatic-heterochromatic boundaries and the locations of repetitive and single-copy DNA sequences and of chromosome-specific BAC clones on chromosomes. The combination of cytogenetic and genetic methods yields a highresolution physical map. FISH allows direct mapping of specific DNA sequences inside the cell (interphase nuclei), along meiotic pachytene chromosomes and isolated chromatin (DNA fibres). The increased sensitivity of the technique and its ability to detect gene locations provide a powerful research tool for genetic and pre-breeding studies. FISH-based physical mapping plays an important role and is increasingly used for studies at the cytological level on the chromatin organization that controls gene expression and regulation. The present minireview describes some of the benefits of alternative FISH-based techniques and their application for studying plant chromosomes and genomes.

Nutritional factors in stroke

Observational studies support the role of modifying lifestyle-related risk factors such as diet, physical activity and alcohol use in stroke prevention. For example, increased Na intake is associated with hypertension, and reduction in salt consumption may significantly lower blood pressure and may reduce stroke mortality. Moderately elevated homocysteine levels may be associated with stroke and are associated with deficiency of dietary intake of folate, vitamin B6 and vitamin B12. Consumption of a diet rich in fruits, vegetables, folate, K, Ca, Mg, dietary fibre, fish and milk may protect against stroke. Regular physical activity may also protect against stroke through its role in controlling various risk factors such as hypertension, diabetes mellitus and obesity. The role of fat intake as a risk factor for stroke remains uncertain, whereas the association between stroke and cholesterol has more convincingly been demonstrated by the recent intervention trials using statins. There is also evidence that a low serum albumin may be causally linked to stroke risk and outcome and that a significant number of stroke patients are undernourished on admission and their nutritional status deteriorates further whilst in hospital. Undernutrition is associated with increasing morbidity and mortality and nutritional supplements may have some beneficial effect on some outcome measures.