Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens.

BACKGROUND: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. RESULT: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. CONCLUSIONS: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.

Hypothalamic distribution, adenohypophyseal receptor expression, and ligand functionality of RFamide-related peptide 3 in the mare during the breeding and nonbreeding seasons.

RFamide-related peptide 3 (RFRP3), the mammalian homologue of avian gonadotropin-inhibitory hormone, has been shown to negatively regulate the secretion of LH and may contribute to reproductive seasonality in some species. Herein, we examined the presence and potential role of the RFRP3-signaling system in regulating LH secretion in the mare during the breeding and nonbreeding seasons. Hypothalamic NPVF mRNA (the precursor mRNA for RFRP3) was detected at the level of the dorsomedial nucleus and paraventricular nucleus, but expression did not change with season. A greater number of RFRP3-expressing cells was observed throughout the rostral-caudal extension of the dorsomedial nucleus. Furthermore, adenohypophyseal expression of the RFRP3 receptor (NPFFR1) during the winter anovulatory season did not differ from that during either the follicular or luteal phases of the estrous cycle. When tested in primary adenohypophyseal cell culture or in vivo during both the breeding and nonbreeding seasons, neither equine nor ovine peptide sequences for RFRP3 suppressed basal or GnRH-mediated release of LH. However, infusion of RF9, an RFRP3 receptor-signaling antagonist, into seasonally anovulatory mares induced a robust increase in secretion of LH both before and following continuous treatment with GnRH. The results indicate that the cellular machinery associated with RFRP3 function is present in the equine hypothalamus and adenohypophysis. However, evidence for functionality of the RFRP3-signaling network was only obvious when an antagonist RF9 was employed. Because GnRH-induced release of LH was not affected by RF9, its actions may occur upstream from the gonadotrope to stimulate or disinhibit secretion of GnRH.

Marinobufagenin regulates permeability and gene expression of brain endothelial cells.

Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1-, 10-, and 100-nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) downregulated concentrations of the soluble VEGFR transcript sFLT by 59% but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1,069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment upregulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment downregulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16, and TMEM207) and cell signaling (GRIN2C, SLC8A1, and ESR1). The level of downregulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while downregulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C, and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.

Alternative parameterizations of relatedness in whole genome association analysis of pre-weaning traits of Nelore-Angus calves.

Gestation length, birth weight, and weaning weight of F2 Nelore-Angus calves (n = 737) with designed extensive full-sibling and half-sibling relatedness were evaluated for association with 34,957 SNP markers. In analyses of birth weight, random relatedness was modeled three ways: 1) none, 2) random animal, pedigree-based relationship matrix, or 3) random animal, genomic relationship matrix. Detected birth weight-SNP associations were 1,200, 735, and 31 for those parameterizations respectively; each additional model refinement removed associations that apparently were a result of the built-in stratification by relatedness. Subsequent analyses of gestation length and weaning weight modeled genomic relatedness; there were 40 and 26 trait-marker associations detected for those traits, respectively. Birth weight associations were on BTA14 except for a single marker on BTA5. Gestation length associations included 37 SNP on BTA21, 2 on BTA27 and one on BTA3. Weaning weight associations were on BTA14 except for a single marker on BTA10. Twenty-one SNP markers on BTA14 were detected in both birth and weaning weight analyses.

Genetic evaluation of crossbred Bos indicus cow temperament at parturition.

Cow temperament at parturition may be mostly a measure of aggressiveness. The heritability of cow temperament at parturition in Bos taurus cows has been reported to be low. The objectives of this study were to estimate the heritability of cow temperament at parturition, conduct a genome-wide association analysis of cow temperament at the time of parturition, and estimate the correspondence of cow temperament at the time of parturition with cow productive performance and early-life temperament traits in Bos indicus crossbreds. Cow temperament was assessed from 1 to 5 indicating increasing levels of aggressiveness of cows (937 cows and 4,337 parturitions) from 2005 to 2022. Estimates of heritability and repeatability were 0.12 ±â€…0.024 and 0.24 ±â€…0.018. The estimates of proportion of phenotypic variance were 0.13 ±â€…0.019 and 0.02 ±â€…0.011 for permanent and maternal permanent environmental components, respectively. Estimates of heritability for maximum lifetime temperament score and proportions of temperament scores >1 were 0.18 ±â€…0.07 and 0.13 ±â€…0.072. Within cycles (generations), 2-yr-old cows had lower temperament score means than cows in most other age categories. There were low to moderate positive estimates of unadjusted correlation coefficients (r = 0.22 to 0.29; P < 0.05) of unadjusted temperament score with temperament measured on the same females when they were 8 mo old. There were low to moderate positive estimates of correlation coefficients (r = 0.09 to 0.37; P < 0.05) of unadjusted temperament score with calving rate, weaning rate, weaning weight per cow exposed, and weaning weight per 454 kg cow weight at weaning. Cows with the lowest temperament score had lower (P < 0.05) calving and weaning rate than cows in other temperament categories. Within 3 of 5 cycles, cows with the lowest temperament score (totally docile) had lower (P < 0.05) weaning weight per cow exposed than cows in other temperament categories. There were 2 SNP on BTA 4 associated with maximum lifetime temperament score (FDR < 0.05). The non-genetic influence of a cow's mother was documented in her own temperament measured at the time of calving; this may be a consequence of learned behavior. Less aggressiveness displayed by cows at the time of calving may be accompanied by lower reproductive and maternal performance.

Cow temperament was evaluated in 1/2 Nellore 1/2 Angus cows from four distinct generations (five herds) from 2005 to 2022. Cows were scored when their calves were processed (1 d age) as 1 = totally docile, 2 = protective, but not aggressive, 3 = moderately aggressive when calf is disturbed, 4 = very aggressive when calf is disturbed, and 5 = very aggressive even when calf is not disturbed. Similar to results in Bos taurus cows, the heritability of this trait was low. The repeatability was more moderate, indicating that additional records from a cow would be beneficial for selection purposes. Young cows had lower scores, indicating more docile behavior. This may be because a strong maternal protective instinct develops and strengthens over time. Temperament measured when cows were 8 mo old was moderately associated with their temperament as mature cows at the time of parturition. Cows with low temperament scores (more docile) had, in several cases, lower reproductive performance and production. Experiential accumulation appears to be important for cow temperament near the time of calving, including the cow's experience as a calf from her dam.

Current challenges and future of agricultural genomes to phenomes in the USA.

Dramatic improvements in measuring genetic variation across agriculturally relevant populations (genomics) must be matched by improvements in identifying and measuring relevant trait variation in such populations across many environments (phenomics). Identifying the most critical opportunities and challenges in genome to phenome (G2P) research is the focus of this paper. Previously (Genome Biol, 23(1):1-11, 2022), we laid out how Agricultural Genome to Phenome Initiative (AG2PI) will coordinate activities with USA federal government agencies expand public-private partnerships, and engage with external stakeholders to achieve a shared vision of future the AG2PI. Acting on this latter step, AG2PI organized the "Thinking Big: Visualizing the Future of AG2PI" two-day workshop held September 9-10, 2022, in Ames, Iowa, co-hosted with the United State Department of Agriculture's National Institute of Food and Agriculture (USDA NIFA). During the meeting, attendees were asked to use their experience and curiosity to review the current status of agricultural genome to phenome (AG2P) work and envision the future of the AG2P field. The topic summaries composing this paper are distilled from two 1.5-h small group discussions. Challenges and solutions identified across multiple topics at the workshop were explored. We end our discussion with a vision for the future of agricultural progress, identifying two areas of innovation needed: (1) innovate in genetic improvement methods development and evaluation and (2) innovate in agricultural research processes to solve societal problems. To address these needs, we then provide six specific goals that we recommend be implemented immediately in support of advancing AG2P research.

Inter-Individual Variation in DNA Methylation Patterns across Two Tissues and Leukocytes in Mature Brahman Cattle.

Quantifying the natural inter-individual variation in DNA methylation patterns is important for identifying its contribution to phenotypic variation, but also for understanding how the environment affects variability, and for incorporation into statistical analyses. The inter-individual variation in DNA methylation patterns in female cattle and the effect that a prenatal stressor has on such variability have yet to be quantified. Thus, the objective of this study was to utilize methylation data from mature Brahman females to quantify the inter-individual variation in DNA methylation. Pregnant Brahman cows were transported for 2 h durations at days 60 ± 5; 80 ± 5; 100 ± 5; 120 ± 5; and 140 ± 5 of gestation. A non-transport group was maintained as a control. Leukocytes, amygdala, and anterior pituitary glands were harvested from eight cows born from the non-transport group (Control) and six from the transport group (PNS) at 5 years of age. The DNA harvested from the anterior pituitary contained the greatest variability in DNA methylation of cytosine-phosphate-guanine (mCpG) sites from both the PNS and Control groups, and the amygdala had the least. Numerous variable mCpG sites were associated with retrotransposable elements and highly repetitive regions of the genome. Some of the genomic features that had high variation in DNA methylation are involved in immune responses, signaling, responses to stimuli, and metabolic processes. The small overlap of highly variable CpG sites and features between tissues and leukocytes supports the role of variable DNA methylation in regulating tissue-specific gene expression. Many of the CpG sites that exhibited high variability in DNA methylation were common between the PNS and Control groups within a tissue, but there was little overlap in genomic features with high variability. The interaction between the prenatal environment and the genome could be responsible for the differences in location of the variable DNA methylation.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice.

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

Skeletal Muscle Expression of Actinin-3 (ACTN3) in Relation to Feed Efficiency Phenotype of F2 Bos indicus - Bos taurus Steers.

In this study, actinin-3 (ACTN3) gene expression was investigated in relation to the feed efficiency phenotype in Bos indicus - Bos taurus crossbred steers. A measure of relative feed efficiency based on residual feed intake relative to predictions from the NRC beef cattle model was analyzed by the use of a mixed linear model that included sire and family nested within sire as fixed effects and age, animal type, sex, condition, and breed as random effects for 173 F2 Nellore-Angus steers. Based on these residual intake observations, individuals were ranked from most efficient to least efficient. Skeletal muscle samples were analyzed from 54 steers in three groups of 18 (high efficiency, low efficiency, and a statistically average group). ACTN3, which encodes a muscle-specific structural protein, was previously identified as a candidate gene from a microarray analysis of RNA extracted from muscle samples obtained from a subset of steers from each of these three efficiency groups. The expression of ACTN3 was evaluated by quantitative reverse transcriptase PCR analysis. The expression of ACTN3 in skeletal muscle was 1.6-fold greater in the inefficient steer group than in the efficient group (p = 0.007). In addition to expression measurements, blocks of SNP haplotypes were assessed for breed or parent of origin effects. A maternal effect was observed for ACTN3 inheritance, indicating that a maternal B. indicus block conferred improved residual feed efficiency relative to the B. taurus copy (p = 0.03). A SNP haplotype analysis was also conducted for m-calpain (CAPN2) and fibronectin 1 (FN1), and a significant breed effect was observed for both genes, with B. indicus and B. taurus alleles each conferring favorable efficiency when inherited maternally (p = 0.03 and p = 0.04). Because the ACTN3 structural protein is specific to fast-twitch (type II) muscle fibers and not present in slow-twitch muscle fibers (type I), muscle samples used for expression analysis were also assayed for fiber type ratio (type II/type I). Inefficient animals had a fast fiber type ratio 1.8-fold greater than the efficient animals (p = 0.027). Because these fiber-types exhibit different metabolic profiles, we hypothesize that animals with a greater proportion of fast-twitch muscle fibers are also less feed efficient.

DNA methylation patterns and gene expression from amygdala tissue of mature Brahman cows exposed to prenatal stress.

Prenatal stress can alter postnatal performance and temperament of cattle. These phenotypic effects may result from changes in gene expression caused by stress-induced epigenetic alterations. Specifically, shifts in gene expression caused by DNA methylation within the brain's amygdala can result in altered behavior because it regulates fear, stress response and aggression in mammals Thus, the objective of this experiment was to identify DNA methylation and gene expression differences in the amygdala tissue of 5-year-old prenatally stressed (PNS) Brahman cows compared to control cows. Pregnant Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 days of gestation. A non-transported group (n = 48) were controls (Control). Amygdala tissue was harvested from 6 PNS and 8 Control cows at 5 years of age. Overall methylation of gene body regions, promoter regions, and cytosine-phosphate-guanine (CpG) islands were compared between the two groups. In total, 202 genes, 134 promoter regions, and 133 CpG islands exhibited differential methylation (FDR ≤ 0.15). Following comparison of gene expression in the amygdala between the PNS and Control cows, 2 differentially expressed genes were identified (FDR ≤ 0.15). The minimal differences observed could be the result of natural changes of DNA methylation and gene expression as an animal ages, or because this degree of transportation stress was not severe enough to cause lasting effects on the offspring. A younger age may be a more appropriate time to assess methylation and gene expression differences produced by prenatal stress.

Complete Whole Genome Sequences of Escherichia coli Surrogate Strains and Comparison of Sequence Methods with Application to the Food Industry.

In 2013, the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) began transitioning to whole genome sequencing (WGS) for foodborne disease outbreak- and recall-associated isolate identification of select bacterial species. While WGS offers greater precision, certain hurdles must be overcome before widespread application within the food industry is plausible. Challenges include diversity of sequencing platform outputs and lack of standardized bioinformatics workflows for data analyses. We sequenced DNA from USDA-FSIS approved, non-pathogenic E. coli surrogates and a derivative group of rifampicin-resistant mutants (rifR) via both Oxford Nanopore MinION and Illumina MiSeq platforms to generate and annotate complete genomes. Genome sequences from each clone were assembled separately so long-read, short-read, and combined sequence assemblies could be directly compared. The combined sequence data approach provides more accurate completed genomes. The genomes from these isolates were verified to lack functional key E. coli elements commonly associated with pathogenesis. Genetic alterations known to confer rifR were also identified. As the food industry adopts WGS within its food safety programs, these data provide completed genomes for commonly used surrogate strains, with a direct comparison of sequence platforms and assembly strategies relevant to research/testing workflows applicable for both processors and regulators.

Genome-wide DNA methylation alteration in prenatally stressed Brahman heifer calves with the advancement of age.

Possible phenotypic impairments associated with maternal stress during gestation in beef cattle may be explained by epigenetic effects. This study examined the impact of prenatal transportation stress on DNA methylation of lymphocytes of Brahman cows over the first 5 years of life. Methylation analysis through reduced representation bisulphite sequencing was conducted on DNA from lymphocytes from 28 paired samples from 6 prenatally stressed (PNS) and 8 control (Control) females obtained initially when they were 28 days of age and 5 years of age. There were 14,386 CpG (C = cytosine; p = phosphate; G = guanine) sites differentially methylated (P < 0.01) in 5-yr-old Control cows compared to their lymphocyte DNA at 28 days of age, this number was slightly decreased in 5-yr-old PNS with 13,378 CpG sites. Only 2,749 age-related differentially methylated CpG sites were seen within PNS females. There were 2,637 CpG sites differentially methylated (P < 0.01) in PNS cows relative to Controls at 5 years of age. There were differentially methylated genes in 5-yr-old cows that contributed similarly to altered gene pathways in both treatment groups. Canonical pathways altered in PNS compared to Control cows at 5 years of age were mostly related to development and growth, nervous system development and function, and immune response. Prenatal stress appeared to alter the epigenome in Brahman cows compared to Control at 5 years of age, which implies a persistent intervention in DNA methylation in lymphocytes, and may confer long-lasting effects on gene expression, and consequently relevant phenotypic changes.

Influence of prenatal transportation stress-induced differential DNA methylation on the physiological control of behavior and stress response in suckling Brahman bull calves.

The objective of this experiment was to examine potential differential methylation of DNA as a mechanism for altered behavioral and stress responses in prenatally stressed (PNS) compared with nonprenatally stressed (Control) young bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation (Transported group) or maintained as nontransported Controls (n = 48). From the offspring born to Transported and Control cows, a subset of 28-d-old intact bulls (n = 7 PNS; n = 7 Control) were evaluated for methylation of DNA of behavior and stress response-associated genes. Methylation of DNA from white blood cells was assessed via reduced representation bisulfite sequencing methods. Because increased methylation of DNA within gene promoter regions has been associated with decreased transcriptional activity of the corresponding gene, differentially methylated (P ≤ 0.05) CG sites (cytosine followed by a guanine nucleotide) located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. Among differentially methylated genes (P ≤ 0.05) related to behavior and the stress response were OPRK1, OPRM1, PENK, POMC, NR3C2, TH, DRD1, DRD5, COMT, HTR6, HTR5A, GABRA4, GABRQ, and GAD2. Among altered (P < 0.05) signaling pathways related to behavior and the stress response were Opioid Signaling, Corticotropin-Releasing Hormone Signaling, Dopamine Receptor Signaling, Dopamine-DARPP32 Feedback in cAMP Signaling, Serotonin Receptor Signaling, and GABA Receptor Signaling. Alterations to behavior and stress response-related genes and canonical pathways supported previously observed elevations in temperament score and serum cortisol through weaning in the larger population of PNS calves from which bulls in this study were derived. Differential methylation of DNA and predicted alterations to behavior and stress response-related pathways in PNS compared with Control bull calves suggest epigenetic programming of behavior and the stress response in utero.

Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation.

Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern preimplantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of preimplantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and preimplantation blastocyst growth and development in sheep.

Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B. bubalis) chromosome 20.

BACKGROUND: Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method. RESULTS: As proof of principle for this dissociation curve method, we generated new maps of river buffalo (Bubalus bubalis) chromosome 20 by both dissociation curve analysis and conventional marker scoring. We also obtained sequence data to augment dissociation curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46%) were suitable as cross-species markers for dissociation curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly. CONCLUSION: Dissociation curve analysis is reliable and efficient for radiation hybrid panel scoring, and is more sensitive and robust than conventional gel-based typing methods. Several markers could be scored only by the new method, and ambiguous scores were reduced. PCR-based dissociation curve analysis decreases both time and resources needed for construction of radiation hybrid panel marker maps and represents a significant improvement over gel-based methods in any species.

A first generation whole genome RH map of the river buffalo with comparison to domestic cattle.

BACKGROUND: The recently constructed river buffalo whole-genome radiation hybrid panel (BBURH5000) has already been used to generate preliminary radiation hybrid (RH) maps for several chromosomes, and buffalo-bovine comparative chromosome maps have been constructed. Here, we present the first-generation whole genome RH map (WG-RH) of the river buffalo generated from cattle-derived markers. The RH maps aligned to bovine genome sequence assembly Btau_4.0, providing valuable comparative mapping information for both species. RESULTS: A total of 3990 markers were typed on the BBURH5000 panel, of which 3072 were cattle derived SNPs. The remaining 918 were classified as cattle sequence tagged site (STS), including coding genes, ESTs, and microsatellites. Average retention frequency per chromosome was 27.3% calculated with 3093 scorable markers distributed in 43 linkage groups covering all autosomes (24) and the X chromosomes at a LOD >or= 8. The estimated total length of the WG-RH map is 36,933 cR5000. Fewer than 15% of the markers (472) could not be placed within any linkage group at a LOD score >or= 8. Linkage group order for each chromosome was determined by incorporation of markers previously assigned by FISH and by alignment with the bovine genome sequence assembly (Btau_4.0). CONCLUSION: We obtained radiation hybrid chromosome maps for the entire river buffalo genome based on cattle-derived markers. The alignments of our RH maps to the current bovine genome sequence assembly (Btau_4.0) indicate regions of possible rearrangements between the chromosomes of both species. The river buffalo represents an important agricultural species whose genetic improvement has lagged behind other species due to limited prior genomic characterization. We present the first-generation RH map which provides a more extensive resource for positional candidate cloning of genes associated with complex traits and also for large-scale physical mapping of the river buffalo genome.

Prenatal transportation stress alters genome-wide DNA methylation in suckling Brahman bull calves.

The objective of this experiment was to identify genome-wide differential methylation of DNA in young prenatally stressed (PNS) bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation or maintained as nontransported Controls (n = 48). Methylation of DNA from white blood cells from a subset of 28-d-old intact male offspring (n = 7 PNS; n = 7 Control) was assessed via reduced representation bisulfite sequencing. Samples from PNS bulls contained 16,128 CG, 226 CHG, and 391 CHH (C = cytosine; G = guanine; H = either adenine, thymine, or cytosine) sites that were differentially methylated compared to samples from Controls. Of the CG sites, 7,407 were hypermethylated (at least 10% more methylated than Controls; P ≤ 0.05) and 8,721 were hypomethylated (at least 10% less methylated than Controls; P ≤ 0.05). Increased DNA methylation in gene promoter regions typically results in decreased transcriptional activity of the region. Therefore, differentially methylated CG sites located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. In PNS bull calves, 113 pathways were altered (P ≤ 0.05) compared to Controls. Among these were pathways related to behavior, stress response, metabolism, immune function, and cell signaling. Genome-wide differential DNA methylation and predicted alterations to pathways in PNS compared with Control bull calves suggest epigenetic programming of biological systems in utero.

The contribution of transposable elements to Bos taurus gene structure.

In an effort to identify the contribution of TEs to bovine genome evolution, the abundance, distribution and insertional orientation of TEs were examined in all bovine nuclear genes identified in sequence build 2.1 (released October 11, 2005). Exons, introns and promoter segments (3 kb upstream the transcription initiation sites) were screened with the RepeatMasker program. Most of the genes analyzed contained TE insertions, with an average of 18 insertions/gene. The majority of TE insertions identified were classified as retrotransposons and the remainder classified as DNA transposons. TEs were inserted into exons and promoter segments infrequently, while insertion into intron sequences was strikingly more abundant. The contribution of TEs to exon sequence is of great interest because TE insertions can directly influence the phenotype by altering protein sequences. We report six cases where the entire exon sequences of bovine genes are apparently derived from TEs and one of them, the insertion of Charlie into a bovine transcript similar to the zinc finger 452 gene is analyzed in detail. The great similarity of the TE-cassette sequence to the ZNF452 protein and phylogenetic relationship strongly suggests the occurrence of Charlie 10 DNA exaptation in the mammalian zinc finger 452 gene.

Ionizing radiation-induced bioeffects in space and strategies to reduce cellular injury and carcinogenesis.

BACKGROUND: The bioeffects of space radiation on organisms outside of the environment of Earth's magnetosphere are a concern for long-duration exploration spaceflights. Potential mutagenic effects from space radiation exposure result from direct DNA damage or indirectly from the production of reactive oxygen species (ROS). HYPOTHESES: 1) Transepithelial electrical resistance (TER) measurements in cell culture monolayers may be used as a model system for detecting cell damage produced by exposure to simulated space radiation and for testing potential chemoprotective agents; 2) biomarkers of exposure that quantitate indirect radiation effects may allow prediction of cellular DNA damage; and 3) a multiple agent, chemoprevention cocktail may reduce the bioeffects of simulated space radiation. METHODS: Normal human and canine lung, breast, and renal epithelial cells were assayed in vitro and exposed to escalating doses of gamma or heavy-ion carbon (290 MeV/u), ceon (400 MeV/u), or iron (600 MeV/u) irradiation. Post-exposure measurements of TER, lipid peroxidation (LP) via measurement of 4-hydroxy-nonenal (4-HNE), and malonaldehyde (MDA) and assessment of chromosome damage via fluorescence in situ hybridization with tandem labeling of chromosome 1 were performed. RESULTS: Cells exposed to intermediate or high doses of radiation (5, 10, and 25 Gy) showed characteristic diminution in TER, thought to be secondary to dysfunction of tight junctions, and associated with membrane LP and other mechanisms. The cells also showed increases in 4-HNE + MDA measurements and increased frequency of chromosomal aberrations. Preliminary studies of cells incubated with media containing a combination of chemoprotective agents at the time of radiation exposure showed a 15-50% reduction in the radiation-induced changes in membrane resistance, levels of LP, and chromosomal aberrations relative to their unprotected cellular counterparts. CONCLUSION: TER measurement, in conjunction with measures of LP, may provide a useful model for determination of physiological changes caused by radiation exposure and the efficacy of chemoprotective agents. A multi-agent mixture of chemoprotective agents may be more effective than previously evaluated single agents alone.