Biological effects of intraoperative radiation therapy: histopathological changes and immunomodulation in breast cancer patients.

Introduction: Intraoperative radiation therapy (IORT) delivers a single accelerated radiation dose to the breast tumor bed during breast-conserving surgery (BCS). The synergistic biologic effects of simultaneous surgery and radiation remain unclear. This study explores the cellular and molecular changes induced by IORT in the tumor microenvironment and its impact on the immune response modulation. Methods: Patients with hormone receptor (HR)-positive/HER2-negative, ductal carcinoma in situ (DCIS), or early-stage invasive breast carcinoma undergoing BCS with margin re-excision were included. Histopathological evaluation and RNA-sequencing in the re-excision tissue were compared between patients with IORT (n=11) vs. non-IORT (n=11). Results: Squamous metaplasia with atypia was exclusively identified in IORT specimens (63.6%, p=0.004), mimicking DCIS. We then identified 1,662 differentially expressed genes (875 upregulated and 787 downregulated) between IORT and non-IORT samples. Gene ontology analyses showed that IORT was associated with the enrichment of several immune response pathways, such as inflammatory response, granulocyte activation, and T-cell activation (p<0.001). When only considering normal tissue from both cohorts, IORT was associated with intrinsic apoptotic signaling, response to gamma radiation, and positive regulation of programmed cell death (p<0.001). Using the xCell algorithm, we inferred a higher abundance of γδ T-cells, dendritic cells, and monocytes in the IORT samples. Conclusion: IORT induces histological changes, including squamous metaplasia with atypia, and elicits molecular alterations associated with immune response and intrinsic apoptotic pathways. The increased abundance of immune-related components in breast tissue exposed to IORT suggests a potential shift towards active immunogenicity, particularly immune-desert tumors like HR-positive/HER2-negative breast cancer.

Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome contributes to matrix metalloprotease (MMP) dysregulation impacting tumor invasion, which is the first step of the metastatic process. METHODS: We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with MMP gene expression and invasion. We employed CRISPR/Cas9 to disrupt the CCCTC-Binding Factor (CTCF) binding site on an insulator element downstream of the MMP8 gene (IE8) in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. The potential of our findings to predict the progression of ductal carcinoma in situ (DCIS), was tested in data from clinical specimens. RESULTS: We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was associated with worse relapse-free (HR = 1.57 [1.06 - 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 - 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability and collagen degradation. We observed that the MMP expression pattern predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01). CONCLUSION: Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

Chronic HCV infection promotes cytotoxicity in antigen-specific CD8+ T cells regardless of virus specificity.

Introduction: Despite advancements in hepatitis C virus (HCV) infection treatment, HCV still represents a significant public health burden. Besides progressive hepatic damage, viral persistence has lasting effects on innate and adaptive immune responses. Lack of a complete understanding of the factors driving an effective HCV response contributes to the failure to develop a vaccine for prevention. This study advances the existing knowledge on HCV-specific CD8+ T cells and describes the impact of current or past HCV infection on CD8+ T cells specific for other viruses. Methods: We used barcoded-dextramers to identify and sort CD8+ T cells specific for HCV, cytomegalovirus, and influenza, and characterized them using single-cell RNA sequencing technology. Our cohort included chronic (cHCV), spontaneously resolved (rHCV), and subjects undergoing direct-acting antiviral (DAA) therapy. Results: We show that HCV-specific CD8+ T cells have cytotoxic features in patients with cHCV, which is progressively reduced with DAA therapy and persists 12 weeks after treatment completion. We also observe a shift in the CD8+ T cell phenotype on DAA treatment, with decreased effector memory and exhausted cell signatures. In rHCV, we also detected a smaller proportion of effector memory cells compared to cHCV. The proportion of CD8+ exhausted T cells in cHCV and rHCV subjects was comparable. Moreover, we also observed that non-HCV virus-specific CD8+ T cells exhibit robust cytotoxic traits during cHCV infection. Discussion: Altogether, our findings suggest that cHCV infection promotes cytotoxicity in CD8+ T cells regardless of virus specificity. The immunological changes caused by cHCV infection in CD8+ T cells may contribute to worsening the ongoing hepatic damage caused by HCV infection or exacerbate the immune response to possible co-infections. Our data provide a resource to groups exploring the underlying mechanisms of HCV-specific T cell spontaneous and treatment-induced resolution to inform the development of effective vaccines against HCV infection.

Age-related next-generation sequencing mutational analysis in 1196 melanomas.

BACKGROUND AND OBJECTIVES: Melanoma mutational burden is high and approximately 50% have oncogenic mutations in BRAF. We sought to evaluate age-related mutational differences in melanoma. METHODS: We analyzed melanoma samples in the Genomics Evidence Neoplasia Information Exchange database. Targetable mutations were identified using the Precision Oncology Knowledge Base (OncoKB). RESULTS: We found 1194 patients with a common set of 30 genes. The top mutated genes in patients <40 years old (y/o) (n = 98) were BRAF (59%), TP53 (31%), NRAS (17%), and PTEN (14%); in 40-59 y/o (n = 354) were BRAF (51%), NRAS (30%), TP53 (26%), and APC (13%); and in ≥60 y/o (n = 742) were BRAF (38%), NRAS (33%), TP53 (26%), and KDR (19%). BRAF mutations were almost mutually exclusive from NRAS mutations in <40 y/o (58/59). Mutational burden increased with age, with means of 2.39, 2.92, and 3.67 mutations per sample in patients <40, 40-59, and ≥60 y/o, respectively (p < 0.0001). There were 10 targetable mutations meeting OncoKB criteria for melanoma: BRAF (level 1), RET (level 1), KIT (level 2), NRAS (level 3A), TP53 (level 3A), and FGFR2, MET, PTEN, PIK3CA, and KRAS (level 4). CONCLUSIONS: Mutations in melanoma have age-related differences and demonstrates potential targetable mutations for personalized therapies.

Is the 21-Gene Recurrence Score on Core Needle Biopsy Equivalent to Surgical Specimen in Early-Stage Breast Cancer? A Comparison of Gene Expression Between Paired Core Needle Biopsy and Surgical Specimens.

BACKGROUND: Molecular testing on surgical specimens predicts disease recurrence and benefit of adjuvant chemotherapy in hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) early-stage breast cancer (EBC). Testing on core biopsies has become common practice despite limited evidence of concordance between core/surgical samples. In this study, we compared the gene expression of the 21 genes and the recurrence score (RS) between paired core/surgical specimens. METHODS: Eighty patients with HR+/HER2- EBC were evaluated from two publicly available gene expression datasets (GSE73235, GSE76728) with paired core/surgical specimens without neoadjuvant systemic therapy. The expression of the 21 genes was compared in paired samples. A microarray-based RS was calculated and a value ≥ 26 was defined as high-RS. The concordance rate and kappa statistic were used to evaluate the agreement between the RS of paired samples. RESULTS: Overall, there was no significant difference and a high correlation in the gene expression levels of the 21 genes between paired samples. However, CD68 and RPLP0 in GSE73235, AURKA, BAG1, and TFRC in GSE76728, and MYLBL2 and ACTB in both datasets exhibited weak to moderate correlation (r < 0.5). There was a high correlation of the microarray-based RS between paired samples in GSE76728 (r = 0.91, 95% confidence interval [CI] 0.81-0.96) and GSE73235 (r = 0.82, 95% CI 0.71-0.89). There were no changes in RS category in GSE76728, whereas 82% of patients remained in the same RS category in GSE73235 (κ = 0.64). CONCLUSIONS: Gene expression levels of the 21-gene RS showed a high correlation between paired specimens. Potential sampling and biological variability on a set of genes need to be considered to better estimate the RS from core needle biopsy.

Clinical Implications of Transcriptomic Changes After Neoadjuvant Chemotherapy in Patients with Triple-Negative Breast Cancer.

BACKGROUND: Pathological response to neoadjuvant chemotherapy (NAC) is critical in prognosis and selection of systemic treatments for patients with triple-negative breast cancer (TNBC). The aim of this study is to identify gene expression-based markers to predict response to NAC. PATIENTS AND METHODS: A survey of 43 publicly available gene expression datasets was performed. We identified a cohort of TNBC patients treated with NAC (n = 708). Gene expression data from different studies were renormalized, and the differences between pretreatment (pre-NAC), on-treatment (post-C1), and surgical (Sx) specimens were evaluated. Euclidean statistical distances were calculated to estimate changes in gene expression patterns induced by NAC. Hierarchical clustering and pathway enrichment analyses were used to characterize relationships between differentially expressed genes and affected gene pathways. Machine learning was employed to refine a gene expression signature with the potential to predict response to NAC. RESULTS: Forty nine genes consistently affected by NAC were involved in enhanced regulation of wound response, chemokine release, cell division, and decreased programmed cell death in residual invasive disease. The statistical distances between pre-NAC and post-C1 significantly predicted pathological complete response [area under the curve (AUC) = 0.75; p = 0.003; 95% confidence interval (CI) 0.58-0.92]. Finally, the expression of CCND1, a cyclin that forms complexes with CDK4/6 to promote the cell cycle, was the most informative feature in pre-NAC biopsies to predict response to NAC. CONCLUSIONS: The results of this study reveal significant transcriptomic changes induced by NAC and suggest that chemotherapy-induced gene expression changes observed early in therapy may be good predictors of response to NAC.

Clinicopathological Features of Triple-Negative Breast Cancer Epigenetic Subtypes.

BACKGROUND/OBJECTIVE: Triple-negative breast cancer (TNBC) is a heterogeneous collection of breast tumors with numerous differences including morphological characteristics, genetic makeup, immune-cell infiltration, and response to systemic therapy. DNA methylation profiling is a robust tool to accurately identify disease-specific subtypes. We aimed to generate an epigenetic subclassification of TNBC tumors (epitypes) with utility for clinical decision-making. METHODS: Genome-wide DNA methylation profiles from TNBC patients generated in the Cancer Genome Atlas project were used to build machine learning-based epigenetic classifiers. Clinical and demographic variables, as well as gene expression and gene mutation data from the same cohort, were integrated to further refine the TNBC epitypes. RESULTS: This analysis indicated the existence of four TNBC epitypes, named as Epi-CL-A, Epi-CL-B, Epi-CL-C, and Epi-CL-D. Patients with Epi-CL-B tumors showed significantly shorter disease-free survival and overall survival [log rank; P = 0.01; hazard ratio (HR) 3.89, 95% confidence interval (CI) 1.3-11.63 and P = 0.003; HR 5.29, 95% CI 1.55-18.18, respectively]. Significant gene expression and mutation differences among the TNBC epitypes suggested alternative pathway activation that could be used as ancillary therapeutic targets. These epigenetic subtypes showed complementarity with the recently described TNBC transcriptomic subtypes. CONCLUSIONS: TNBC epigenetic subtypes exhibit significant clinical and molecular differences. The links between genetic make-up, gene expression programs, and epigenetic subtypes open new avenues in the development of laboratory tests to more efficiently stratify TNBC patients, helping optimize tailored treatment approaches.

Evolution of the Mutational Process under Relaxed Selection in Caenorhabditis elegans.

The mutational process varies at many levels, from within genomes to among taxa. Many mechanisms have been linked to variation in mutation, but understanding of the evolution of the mutational process is rudimentary. Physiological condition is often implicated as a source of variation in microbial mutation rate and may contribute to mutation rate variation in multicellular organisms.Deleterious mutations are an ubiquitous source of variation in condition. We test the hypothesis that the mutational process depends on the underlying mutation load in two groups of Caenorhabditis elegans mutation accumulation (MA) lines that differ in their starting mutation loads. "First-order MA" (O1MA) lines maintained under minimal selection for ∼250 generations were divided into high-fitness and low-fitness groups and sets of "second-order MA" (O2MA) lines derived from each O1MA line were maintained for ∼150 additional generations. Genomes of 48 O2MA lines and their progenitors were sequenced. There is significant variation among O2MA lines in base-substitution rate (µbs), but no effect of initial fitness; the indel rate is greater in high-fitness O2MA lines. Overall, µbs is positively correlated with recombination and proximity to short tandem repeats and negatively correlated with 10 bp and 1 kb GC content. However, probability of mutation is sufficiently predicted by the three-nucleotide motif alone. Approximately 90% of the variance in standing nucleotide variation is explained by mutability. Total mutation rate increased in the O2MA lines, as predicted by the "drift barrier" model of mutation rate evolution. These data, combined with experimental estimates of fitness, suggest that epistasis is synergistic.

Early Loss of Histone H2B Monoubiquitylation Alters Chromatin Accessibility and Activates Key Immune Pathways That Facilitate Progression of Ovarian Cancer.

Recent insights supporting the fallopian tube epithelium (FTE) and serous tubal intraepithelial carcinomas (STIC) as the tissue of origin and the precursor lesion, respectively, for the majority of high-grade serous ovarian carcinomas (HGSOC) provide the necessary context to study the mechanisms that drive the development and progression of HGSOC. Here, we investigate the role of the E3 ubiquitin ligase RNF20 and histone H2B monoubiquitylation (H2Bub1) in serous tumorigenesis and report that heterozygous loss of RNF20 defines the majority of HGSOC tumors. At the protein level, H2Bub1 was lost or downregulated in a large proportion of STIC and invasive HGSOC tumors, implicating RNF20/H2Bub1 loss as an early event in the development of serous ovarian carcinoma. Knockdown of RNF20, with concomitant loss of H2Bub1, was sufficient to enhance cell migration and clonogenic growth of FTE cells. To investigate the mechanisms underlying these effects, we performed ATAC-seq and RNA-seq in RNF20 knockdown FTE cell lines. Loss of RNF20 and H2Bub1 was associated with a more open chromatin conformation, leading to upregulation of immune signaling pathways, including IL6. IL6 was one of the key cytokines significantly upregulated in RNF20- and H2Bub1-depleted FTE cells and imparted upon these cells an enhanced migratory phenotype. These studies provide mechanistic insight into the observed oncogenic phenotypes triggered by the early loss of H2Bub1. SIGNIFICANCE: Loss of RNF20 and H2Bub1 contributes to transformation of the fallopian tube epithelium and plays a role in the initiation and progression of high-grade serous ovarian cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/760/F1.large.jpg.

The Epigenomic Landscape of Pituitary Adenomas Reveals Specific Alterations and Differentiates Among Acromegaly, Cushing's Disease and Endocrine-Inactive Subtypes.

Purpose: Pituitary adenomas are one of the most common benign neoplasms of the central nervous system. Although emerging evidence suggests roles for both genetic and epigenetic factors in tumorigenesis, the degree to which these factors contribute to disease remains poorly understood.Experimental Design: A multiplatform analysis was performed to identify the genomic and epigenomic underpinnings of disease among the three major subtypes of surgically resected pituitary adenomas in 48 patients: growth hormone (GH)-secreting (n = 17), adrenocorticotropic hormone (ACTH)-secreting (n = 13, including 3 silent-ACTH adenomas), and endocrine-inactive (n = 18). Whole-exome sequencing was used to profile the somatic mutational landscape, whole-transcriptome sequencing was used to identify disease-specific patterns of gene expression, and array-based DNA methylation profiling was used to examine genome-wide patterns of DNA methylation.Results: Recurrent single-nucleotide and small indel somatic mutations were infrequent among the three adenoma subtypes. However, somatic copy-number alterations (SCNA) were identified in all three pituitary adenoma subtypes. Methylation analysis revealed adenoma subtype-specific DNA methylation profiles, with GH-secreting adenomas being dominated by hypomethylated sites. Likewise, gene-expression patterns revealed adenoma subtype-specific profiles. Integrating DNA methylation and gene-expression data revealed that hypomethylation of promoter regions are related with increased expression of GH1 and SSTR5 genes in GH-secreting adenomas and POMC gene in ACTH-secreting adenomas. Finally, multispectral IHC staining of immune-related proteins showed abundant expression of PD-L1 among all three adenoma subtypes.Conclusions: Taken together, these data stress the contribution of epigenomic alterations to disease-specific etiology among adenoma subtypes and highlight potential targets for future immunotherapy-based treatments. This article reveals novel insights into the epigenomics underlying pituitary adenomas and highlights how differences in epigenomic states are related to important transcriptome alterations that define adenoma subtypes. Clin Cancer Res; 24(17); 4126-36. ©2018 AACR.

Comparative High-Density Linkage Mapping Reveals Conserved Genome Structure but Variation in Levels of Heterochiasmy and Location of Recombination Cold Spots in the Common Frog.

By combining 7077 SNPs and 61 microsatellites, we present the first linkage map for some of the early diverged lineages of the common frog, Rana temporaria, and the densest linkage map to date for this species. We found high homology with the published linkage maps of the Eastern and Western lineages but with differences in the order of some markers. Homology was also strong with the genome of the Tibetan frog Nanorana parkeri and we found high synteny with the clawed frog Xenopus tropicalis We confirmed marked heterochiasmy between sexes and detected nonrecombining regions in several groups of the male linkage map. Contrary to the expectations set by the male heterogamety of the common frog, we did not find male heterozygosity excess in the chromosome previously shown to be linked to sex determination. Finally, we found blocks of loci showing strong transmission ratio distortion. These distorted genomic regions might be related to genetic incompatibilities between the parental populations, and are promising candidates for further investigation into the genetic basis of speciation and adaptation in the common frog.

The red death meets the abdominal bristle: polygenic mutation for susceptibility to a bacterial pathogen in Caenorhabditis elegans.

Understanding the genetic basis of susceptibility to pathogens is an important goal of medicine and of evolutionary biology. A key first step toward understanding the genetics and evolution of any phenotypic trait is characterizing the role of mutation. However, the rate at which mutation introduces genetic variance for pathogen susceptibility in any organism is essentially unknown. Here, we quantify the per-generation input of genetic variance by mutation (VM) for susceptibility of Caenorhabditis elegans to the pathogenic bacterium Pseudomonas aeruginosa (defined as the median time of death, LT50). VM for LT50 is slightly less than VM for a variety of life-history and morphological traits in this strain of C. elegans, but is well within the range of reported values in a variety of organisms. Mean LT50 did not change significantly over 250 generations of mutation accumulation. Comparison of VM to the standing genetic variance (VG) implies a strength of selection against new mutations of a few tenths of a percent. These results suggest that the substantial standing genetic variation for susceptibility of C. elegans to P. aeruginosa can be explained by polygenic mutation coupled with purifying selection.

Temperature, stress and spontaneous mutation in Caenorhabditis briggsae and Caenorhabditis elegans.

Mutation rate often increases with environmental temperature, but establishing causality is complicated. Asymmetry between physiological stress and deviation from the optimal temperature means that temperature and stress are often confounded. We allowed mutations to accumulate in two species of Caenorhabditis for approximately 100 generations at 18°C and for approximately 165 generations at 26°C; 26°C is stressful for Caenorhabditis elegans but not for Caenorhabditis briggsae. We report mutation rates at a set of microsatellite loci and estimates of the per-generation decay of fitness (ΔM(w)), the genomic mutation rate for fitness (U) and the average effect of a new mutation (E[a]), assayed at both temperatures. In C. elegans, the microsatellite mutation rate is significantly greater at 26°C than at 18°C whereas in C. briggsae there is only a slight, non-significant increase in mutation rate at 26°C, consistent with stress-dependent mutation in C. elegans. The fitness data from both species qualitatively reinforce the microsatellite results. The fitness results of C. elegans are potentially complicated by selection but also suggest temperature-dependent mutation; the difference between the two species suggests that physiological stress plays a significant role in the mutational process.

Invariance (?) of mutational parameters for relative fitness over 400 generations of mutation accumulation in Caenorhabditis elegans.

Evidence is accumulating that individuals in poor physiologic condition may accumulate mutational damage faster than individuals in good condition. If poor condition results from pre-existing deleterious mutations, the result is "fitness-dependent mutation rate," which has interesting theoretical implications. Here we report a study in which 10 mutation accumulation (MA) lines of the nematode Caenorhabditis elegans that had previously accumulated mutations for 250 generations under relaxed selection were expanded into sets of "second-order" MA lines and allowed to accumulate mutations for an additional 150 generations. The 10 lines were chosen on the basis of the relative change in fitness over the first 250 generations of MA, five high-fitness lines and five low-fitness lines. On average, the mutational properties (per-generation change in mean relative fitness, mutational variance, and Bateman-Mukai estimates of genomic mutation rate and average mutational effect) of the high-fitness and low-fitness did not differ significantly, and averaged over all lines, the point estimates were extremely close to those of the first-order MA experiment after 200 generations of MA. However, several nonsignificant trends indicate that low-fitness lines may in fact be more likely to suffer mutational damage than high-fitness lines.

Recombination rate between sex chromosomes depends on phenotypic sex in the common frog.

We show that the recombination rate between the sex chromosomes is controlled by phenotypic, rather than genotypic, sex in sex-reversed common frogs. This supports the recent hypothesis that in ectothermic vertebrates sex reversal can prevent the progressive accumulation of mutations to nonrecombining Y chromosomes and their subsequent evolutionary decay.

Sex reversal and primary sex ratios in the common frog (Rana temporaria).

Sex reversal has been suggested to have profound implications for the evolution of sex chromosomes and population dynamics in ectotherms. Occasional sex reversal of genetic males has been hypothesized to prevent the evolutionary decay of nonrecombining Y chromosomes caused by the accumulation of deleterious mutations. At the same time, sex reversals can have a negative effect on population growth rate. Here, we studied phenotypic and genotypic sex in the common frog (Rana temporaria) in a subarctic environment, where strongly female-biased sex ratios have raised the possibility of frequent sex reversals. We developed two novel sex-linked microsatellite markers for the species and used them with a third, existing marker and a Bayesian modelling approach to study the occurrence of sex reversal and to determine primary sex ratios in egg clutches. Our results show that a significant proportion (0.09, 95% credible interval: 0.04-0.18) of adults that were genetically female expressed the male phenotype, but there was no evidence of sex reversal of genetic males that is required for counteracting the degeneration of Y chromosome. The primary sex ratios were mostly equal, but three clutches consisted only of genetic females and three others had a significant female bias. Reproduction of the sex-reversed genetic females appears to create all-female clutches potentially skewing the population level adult sex-ratio consistent with field observations. However, based on a simulation model, such a bias is expected to be small and transient and thus does not fully explain the observed female-bias in the field.

Isolation and characterization of 145 polymorphic microsatellite loci for the common frog (Rana temporaria).

We describe primers and polymerase chain reaction conditions to amplify 145 di-, tri- and tetranucleotide microsatellite loci from the common frog (Rana temporaria), a species commonly used as a model in ecological and evolutionary research. Primers were tested on 46 individuals from two Fennoscandian populations and yielded an average of six to nine alleles per locus (range = 1-30) depending on the population. Average observed heterozygosities in the two populations were 0.16 (range = 0-0.91) and 0.36 (range = 0-1).

Disentangling genetic vs. environmental causes of sex determination in the common frog, Rana temporaria.

BACKGROUND: Understanding of sex ratio dynamics in a given species requires understanding its sex determination system, as well as access for reliable tools for sex identification at different life stages. As in the case of many other amphibians, the common frogs (Rana temporaria) do not have well differentiated sex chromosomes, and an identification of individuals' genetic sex may be complicated by sex reversals. Here, we report results of studies shedding light on the sex determination system and sex ratio variation in this species. RESULTS: A microsatellite locus RtSB03 was found to be sex-linked in four geographically disparate populations, suggesting male heterogamy in common frogs. However, in three other populations examined, no or little evidence for sex-linkage was detected suggesting either ongoing/recent recombination events, and/or frequent sex-reversals. Comparison of inheritance patterns of alleles in RtSB03 and phenotypic sex within sibships revealed a mixed evidence for sex-linkage: all individuals with male phenotype carried a male specific allele in one population, whereas results were more mixed in another population. CONCLUSION: These results make sense only if we assume that the RtSB03 locus is linked to male sex determination factor in some, but not in all common frog populations, and if phenotypic sex-reversals - for which there is earlier evidence from this species - are frequently occurring.

Evidence for multiple retroposition events and gene evolution in the ADP/ATP translocase gene family in Ranid frogs.

Analysis of partial ADP/ATP Translocase gene (Aat) sequences from 11 species of Ranid frogs (Amphibia: Ranidae) identified multiple Aat gene copies. All frog species examined had at least one of the 2 Aat gene copies differing mainly by presence or absence of an approximately 85-bp intron sequence. The sequence data suggest that the gene variant with intron is the ancestral Aat form, whereas the other variants are retroposed copies. Surprisingly, a phylogenetic analysis suggests that multiple reptroposition events have taken place in the Ranid frogs. An analysis including sequences from Drosophila, zebra fish, Xenopus, mouse, and human indicated that all the frog Aat sequences form a monophyletic group with mammalian X-chromosomal Ant2 as a sister unit. Furthermore, the selection test suggested that different variants of the Aat gene appear to have evolved under different modes of evolution (viz., neutral, purifying, and positive selection), and the evolutionary history of different AAT variants appears to differ even among different species.

Genome size variation in the common frog Rana temporaria.

Genome size variation in the common frog (Rana temporaria) was investigated with flow cytometry in three latitudinally separated populations in Sweden to see whether it could provide a useful tool for sex-identification in this species. Depending on the sex and population, per cell DNA content (2C value) varied from 8.823 to 11.266 pg with a mean (+/- SE) 2C value of 9.961+/-0.083 pg. Analysis of variance revealed significant differences in genome size among populations and between sexes. Females had ca 3% larger genomes (x=10.133+/-0.068 pg) than males (x=9.832+/-0.068 pg) in all of the populations (sex x population interaction: P>0.10). Individuals from the southern-most population had significantly (x=9.330+/-0.081 pg) smaller genomes than those from the more northern populations (x=10.032+/-0.085 and x=10.584+/-0.085 pg, respectively). These results are in line with the interpretation that males in the common frog are the heterogametic sex, and that there exists large (up to 12%) geographic variation in genome size in this species. However, the sex differences in the genome size are too small to be useful in individual sex identification.