Development and Application of EST-SSR Markers in Cephalotaxus oliveri From Transcriptome Sequences

Cephalotaxus oliveri is an endemic conifer of China, which has medicinal and ornamental value. However, the limited molecular markers and genetic information are insufficient for further genetic studies of this species. In this study, we characterized and developed the EST-SSRs from transcriptome sequences for the first time. The results showed that a total of 5089 SSRs were identified from 36446 unigenes with a density of one SSR per 11.1 kb. The most common type was trinucleotide repeats, excluding mononucleotide repeats, followed by dinucleotide repeats. AAG/CTT and AT/AT exhibited the highest frequency in the trinucleotide and dinucleotide repeats, respectively. Of the identified SSRs, 671, 1125, and 1958 SSRs were located in CDS, 3′UTR, and 5′UTR, respectively. Functional annotation showed that the SSR-containing unigenes were involved in growth and development with various biological functions. Among successfully designed primer pairs, 238 primer pairs were randomly selected for amplification and validation of EST-SSR markers and 47 primer pairs were identified as polymorphic. Finally, 28 high-polymorphic primers were used for genetic analysis and revealed a moderate level of genetic diversity. Seven natural C. oliveri sampling sites were divided into two genetic groups. Furthermore, the 28 EST-SSRs had 96.43, 71.43, and 78.57% of transferability rate in Cephalotaxus fortune, Ametotaxus argotaenia, and Pseudotaxus chienii, respectively. These markers developed in this study lay the foundation for further genetic and adaptive evolution studies in C. oliveri and related species.

Restarted replication forks are error-prone and cause CAG repeat expansions and contractions

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2–7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

CAG polymorphism of the Androgen Receptor gene and semen parameters in pathozoospermic patients with and without Y chromosome microdeletions, and in normozoospermic men

Introduction. The effect of polymorphic variants of the androgen receptor gene (AR) on spermatogenesis and semen parameters in men with different genotypes for other loci has not been sufficiently studied.The aim of this work was to study the effect of the (CAG)n polymorphism of the AR gene on semen parameters in men with impaired fertility, with and without partial deletions of the AZFс region from the Y chromosome.Materials and methods. The study included 988 unrelated Russian patients with pathozoospermia, including 591 patients without Y chromosome microdeletions and 397 patients with partial deletions of the AZFc region of the Y chromosome. The control group consisted of 131 normozoospermic men. All men who participated in the study underwent semen analysis and genetic testing. Genomic DNA was isolated from peripheral venous blood lymphocytes and ejaculate. The analysis of the polymorphism of (GAG)n repeat in exon 1 of the AR gene was performed using a polymerase chain reaction by the amplified fragment length polymorphism method.Results. Three groups were studied: patients with pathozoospermia with (n = 32) and without (n = 541) Y chromosome microdeletions, and normozoospermic men (control, n = 131). The median and quartiles of the number of CAG repeats in the groups were 22 and 20-25, respectively. According to the number of trinucleotide repeats of the AR gene, all patients were divided into subgroups: carriers of short ((GAG)n ≤18), medium ((GAG)n = 19-25) and long ((GAG)n ≥26) alleles. Medium alleles prevailed in all groups; in men without AZFc deletions and with microdeletions, their frequency was 79.3 and 81.4 %, respectively, in controls - 81.7 %.Conclusion. No correlation was found in examined cohort for semen parameters (sperm concentration and total number, number of live, progressively motile and morphologically normal spermatozoa) from the number of trinucleotide repeats. However, a statistically significant difference (p ≤0.045; FDR correction) was found in concentration and total number, number of live, progressively motile and morphologically normal spermatozoa when comparing men with nomrozoospermia (control) with patients with pathozoospermia with and without partial AZFc deletions in subgroups of carriers of short, medium and long alleles.

Unexpected Associations between the Number of FRAXE Repeats in Boys and Evidence of Diabetes in Their Mothers and Maternal Grandmothers

The FRAXE section of the FMR2 gene, located on the X chromosome, contains varying numbers of trinucleotide repeats; boys with over 200 repeats tend to have mild cognitive impairments, though this is rare. Little is known, however, concerning the phenotypes of individuals with smaller numbers of repeats. Here we answer the research question as to whether the health of ancestors of boys from whom the relevant X chromosome was inherited differed in any way according to the number of FRAXE repeats. Numbers of FRAXE repeats in 5057 boys from the Avon Longitudinal Study of Parents and Children (ALSPAC) were assessed. The distribution was bimodal, with the second smaller distribution starting at 22 repeats. We tested whether possession of 22+ repeats was associated with differences in the health of mothers (who share the X chromosome) and maternal grandmothers (half of whom share it). Female ancestors of boys with >21 repeats compared with <22 showed that maternal grandmothers (MGM) and mothers (M) had an increased risk of diabetes: MGM Type I odds ratio (OR) 2.40 [95%CI: 1.07,5.38]; MGM Type II OR 1.61 [0.96,2.70]; M OR 1.95 [0.96,3.94] using self-reported questionnaire measures. These results were confirmed from maternal medical records which revealed an increased level of diabetes [OR 2.40 (1.16,4.96)] and an increased risk of repeated glycosuria during pregnancy [OR 1.60 (1.08,2.36)]. We tested numbers of FRAXA repeats and showed no such associations, indicating that the findings were not associated with triploid repeats in general. If these findings are replicated elsewhere, there are at least three possible interpretations: (i) maternal diabetes/prediabetes results in an increased number of FRAXE repeats; (ii) women with high numbers of FRAXE repeats are at increased risk of diabetes; or (iii) some common factor, e.g. genomic instability, results in both diabetes and increased repeats.

Molecular Mechanisms of Polyglutamine Pathology and Lessons Learned from Huntington’s Disease

Identification of polymorphic repeating units on DNA as a cause of many neurological disorders has introduced a new concept in molecular biology: Dynamic mutations. Many of the identified dynamic mutations involve expansion of trinucleotide repeats within disease genes. Nine neurodegenerative disorders are currently known to be caused by expanding CAG trinucleotide repeats. These are Huntington’s Disease (HD), Dentato-Rubral Pallidoluysian Atrophy (DRPLA), Spinal and Bulbar Muscular Atrophy (SBMA), and Spinocerebellar Ataxia (SCA) Type 1, 2, 3, 6, 7 and 17. All are inherited in an autosomal dominant fashion except for SBMA, which is X-linked recessive. In all polyQ diseases, the disease mutation involves an increase in the number of CAG repeats within the coding regions of the respective genes. Since CAG triplets encode glutamine in the proteins, diseases caused by CAG repeat expansions are known as “Polyglutamine (polyQ) Diseases”. PolyQ diseases share certain clinical, neuropathological and molecular findings. The most widely studied polyQ disease is HD. In HD and other polyQ diseases, conformational change in the mutant protein causes abnormal folding and proteolysis of the protein, leading to the formation of a toxic polyQ fragment, which aggregates and causes neuronal dysfunction and selective neuronal death in the brain.

Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats

Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington’s Disease. Repeat instability is thought to occur via the formation of intrastrand hairpins during replication, repair, recombination, and transcription though relatively little is known about their structure and dynamics. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 3WJs that only have repeats in the slip-out show two-state behavior, which we attribute to conformational flexibility at the 3WJ branchpoint. When the triplet repeats extend into the adjacent duplex, additional dynamics are observed, which we assign to interconversion of positional isomers. We propose a branchpoint migration model that involves conformational rearrangement, strand exchange, and bulge-loop movement. This migration has implications for how repeat slip-outs are processed by the cellular machinery, disease progression, and their development as drug targets.