Molecular analysis of S-haplotypes in peach, a self-compatible Prunus species

The most commercially grown peach [Prunus persica (L.) Batsch.] cultivars do not require cross-pollination for reasonable fruit set; however, self-incompatibility is a well-known feature within the Prunoideae subfamily. Isoelectric focusing and native polyacrylamide gel electrophoresis of S-ribonucleases; PCR analyses of S-RNase and S-haplotype-specific F-box genes as well as DNA sequencing were carried out to survey the self- (in)compatibility allele pool and to uncover the nature of self-compatibility in peach. From 25 cultivars and hybrids with considerable diversity in phenotype and origin, only two S-haplotypes were detected. Allele identity could be checked by exact length determination of the PCR-amplified fragments and/or partial sequencing of the peach S-1-, S-2-, and Prunus davidiana (Carr.) Franch. S-1 RNases. S-RNases of peach were detected to possess ribonuclease activity, and a single nucleotide polymorphism in the S,-RNase was shown, which represents a synonymous substitution and does not change the amino acid present at the position in the protein. A 700-bp fragment of the peach SFB gene was PCR-amplified, which is similar to the fragment size of functional Prunus L. SFBs. All data obtained in this study may support the contribution of genes outside the S-locus to the self-compatible phenotype of peaches

Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA).

The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation

Reciprocal knock-in mice to investigate the functional redundancy of lamin B1 and lamin B2.

Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated "reciprocal knock-in mice"-mice that make lamin B2 from the Lmnb1 locus (Lmnb1(B2/B2)) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2(B1/B1)). Lmnb1(B2/B2) mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1(B2/B2) mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2(B1/B1) mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance

Background: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology. Results: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0–3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7. Conclusions: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates

Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts)

Apolipoprotein E (apoE) is a multifunctional circulating 34-kDa protein, whose gene encodes single-nucleotide polymorphisms linked to several neurodegenerative diseases. Here, we evaluate whether synthetic RNA/DNA oligonucleoticles (chimeraplasts) can convert a dysfunctional gene, APOE4 (C -> T, Cys112Arg), a risk factor for Alzheimer's disease and other neurological disorders, into wild-type APOE3. In preliminary experiments, we treated recombinant Chinese hamster ovary (CHO) cells stably secreting apoE4 and lymphocytes from a patient homozygous for the epsilon 4 allele with a 68-mer apoE4-to-apoE3 chimeraplast, complexed to the cationic delivery reagent, polyethyleneimine. Genotypes were analyzed after 48 h by routine polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and by genomic sequencing. Clear conversions of APOE4 to APOE3 were detected using either technique, although high concentrations of chimeraplast were needed (>= 800 nM). Spiking experiments of PCR reactions or CHO-K1 cells with the chimeraplast confirmed that the repair was not artifactual. However, when treated recombinant CHO cells were passaged for 10 d and then subcloned, no conversion could be detected when > 90 clones were analyzed by locus-specific PCR-RFLP. We conclude that the apparent efficient repair of the APOE4 gene in CHO cells or lymphocytes 48 h post-treatment is unstable, possibly because the high levels of chimeraplast and polyethyleneimine that were needed to induce nucleotide substitution are cytotoxic

Disease progression in Plasmodium knowlesi malaria is linked to variation in invasion gene family members.

Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥ 0.34, p =  <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region

Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

BAckground: Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. Results: We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. Conclusion: We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material

Transcript-indexed ATAC-seq for precision immune profiling.

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of&nbsp;cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy