Computational identification of microRNA gene loci and precursor microRNA sequences in CHO cell lines

MicroRNAs (miRNAs) have recently entered Chinese hamster ovary (CHO) cell culture technology, due to their severe impact on the regulation of cellular phenotypes. Applications of miRNAs that are envisioned range from biomarkers of favorable phenotypes to cell engineering targets. These applications, however, require a profound knowledge of miRNA sequences and their genomic organization, which exceeds the currently available information of ~400 conserved mature CHO miRNA sequences. Based on these recently published sequences and two independent CHO-K1 genome assemblies, this publication describes the computational identification of CHO miRNA genomic loci. Using BLAST alignment, 415 previously reported CHO miRNAs were mapped to the reference genomes, and subsequently assigned to a distinct genomic miRNA locus. Sequences of the respective precursor-miRNAs were extracted from both reference genomes, folded in silico to verify correct structures and cross-compared. In the end, 212 genomic loci and pre-miRNA sequences representing 319 expressed mature miRNAs (approximately 50% of miRNAs represented matching pairs of 5' and 3' miRNAs) were submitted to the miRBase miRNA repository. As a proof-of-principle for the usability of the published genomic loci, four likely polycistronic miRNA cluster were chosen for PCR amplification using CHO-K1 and DHFR (-) genomic DNA. Overall, these data on the genomic context of miRNA expression in CHO will simplify the development of tools employing stable overexpression or deletion of miRNAs, allow the identification of miRNA promoters and improve detection methods such as microarrays.     

Physical mapping of the IGF2 locus in the South American opossum Monodelphis domestica

The South American opossum Monodelphis domestica has been a model organism for marsupials for many years and has recently been the subject of a large-scale genome sequencing effort that will provide the foundation for comparative studies of gene function and regulation. Genomic imprinting is one mechanism of gene regulation that has received increasing attention due to the impact of imprinting defects on development and disease. We have mapped the imprinted insulin-like growth factor II (IGF2) gene of M. domestica as a first step in understanding the regulatory mechanisms involved in genomic imprinting in this marsupial.     

OMiR: Identification of associations between OMIM diseases and microRNAs

A large number of loci for genetic diseases have been mapped on the human genome and a group of hereditary diseases among them have thus far proven unsuccessful to clone. It is conceivable that such "unclonable" diseases are not linked to abnormalities of protein coding genes (PCGs), but of non-coding RNAs (ncRNAs). We developed a novel approach termed OMiR (OMIM and miRNAs), to test whether microRNAs (miRNAs) exhibit any associations with mapped genetic diseases not yet associated with a PCG. We found that "orphan" genetic disease loci were proximal to miRNA loci more frequently than to loci for which the responsible protein coding gene is known, thus suggesting that miRNAs might be the elusive culprits. Our findings indicate that inclusion of miRNAs among the candidate genes to be considered could assist geneticists in their hunt for disease genes, particularly in the case of rare diseases.     

Thermogenic capabilities of the opossum Monodelphis domestica when warm and cold acclimated: similarities between American and Australian marsupials

1. Monodelphis domestica is a small marsupial mammal from South America. Its thermogenic abilities in the cold were determined when the opossums were both warm (WA) and cold (CA) acclimated. Maximum heat production of M. domestica was obtained at low temperatures in helium-oxygen. 2. Basal metabolic rate (BMR) in the WA animals was 3.2 W/kg and mean body temperature was 32.6 degrees C at 30 degrees C. These values were lower than those generally reported for marsupials. Nevertheless, these M. domestica showed considerable metabolic expansibility in response to cold. Sustained (summit) metabolism was 8-9 times BMR, while peak metabolism was 11-13 times BMR. These maximum values were equal to, or above, those expected in small placentals. 3. Cold acclimation altered the thermal responses of M. domestica, particularly in warm TaS. However, summit metabolism was not significantly increased; nor did M. domestica show a significant thermogenic response to noradrenaline, which in many small placentals elicits non-shivering thermogenesis. The thermoregulatory responses of this American marsupial were, in most aspects, similar to those of Australian marsupials. This suggests that the considerable thermoregulatory abilities of marsupials are of some antiquity.     

First-generation linkage map of the gray, short-tailed opossum, Monodelphis domestica, reveals genome-wide reduction in female recombination rates

The gray, short-tailed opossum, Monodelphis domestica, is the most extensively used, laboratory-bred marsupial resource for basic biologic and biomedical research worldwide. To enhance the research utility of this species, we are building a linkage map, using both anonymous markers and functional gene loci, that will enable the localization of quantitative trait loci (QTL) and provide comparative information regarding the evolution of mammalian and other vertebrate genomes. The current map is composed of 83 loci distributed among eight autosomal linkage groups and the X chromosome. The autosomal linkage groups appear to encompass a very large portion of the genome, yet span a sex-average distance of only 633.0 cM, making this the most compact linkage map known among vertebrates. Most surprising, the male map is much larger than the female map (884.6 cM vs. 443.1 cM), a pattern contrary to that in eutherian mammals and other vertebrates. The finding of genome-wide reduction in female recombination in M. domestica, coupled with recombination data from two other, distantly related marsupial species, suggests that reduced female recombination might be a widespread metatherian attribute. We discuss possible explanations for reduced female recombination in marsupials as a consequence of the metatherian characteristic of determinate paternal X chromosome inactivation.     

Development, inheritance, and linkage-group assignment of 60 novel microsatellite markers for the gray, short-tailed opossum Monodelphis domestica.

Short-tandem-repeat (SSR) or microsatellite polymorphisms are some of the most extensively employed genetic markers in contemporary linkage mapping studies. To date, only a limited number of microsatellites have been isolated in the gray, short-tailed opossum Monodelphis domestica, a South American marsupial widely used for comparative biological and biomedical research. To increase the number of potentially useful mapping markers, we screened 2 microsatellite-enriched genomic libraries containing alternatively (CA)n or (GA)n repeats. A total of 184 clones were sequenced, from which 60 polymorphic microsatellite markers were successfully optimized. The efficiency of this enrichment protocol for M. domestica microsatellite isolation is discussed, and suggestions to improve the outcome are made. All 60 loci showed high allelic diversity, with allele numbers ranging from 2 to 10 in a subset of 33 unrelated animals. Normal Mendelian inheritance was confirmed for all loci by analyzing allelic segregation in 5 two-generation families. One microsatellite appeared to be X linked, and null alleles were found in 5 others. Two-point linkage analyses were implemented using the data on the 5 families, leading to the assignment of 59 of these loci to the existing linkage groups. The 60 novel microsatellites developed in this study will contribute significantly to the M. domestica linkage map, and further QTL mapping studies.     

Linkage mapping and physical localization of the major histocompatibility complex region of the marsupial Monodelphis domestica

We used genetic linkage mapping and fluorescence in situ hybridization (FISH) to conduct the first analysis of genic organization and chromosome localization of the major histocompatibility complex (MHC) of a marsupial, the gray, short-tailed opossum Monodelphis domestica. Family based linkage analyses of two M. domestica MHC Class I genes (UA1, UG) and three MHC Class II genes (DAB, DMA, and DMB) revealed that these genes were tightly linked and positioned in the central region of linkage group 3 (LG3). This cluster of MHC genes was physically mapped to the centromeric region of chromosome 2q by FISH using a BAC clone containing the UA1 gene. An interesting finding from the linkage analyses is that sex-specific recombination rates were virtually identical within the MHC region. This stands in stark contrast to the genome-wide situation, wherein males exhibit approximately twice as much recombination as females, and could have evolutionary implications for maintaining equality between males and females in the ability to generate haplotype diversity in this region. These analyses also showed that three non-MHC genes that flank the MHC region on human chromosome 6, myelin oligodendrocyte glycoprotein (MOG), bone morphogenetic protein 6 (BMP6), and prolactin (PRL), are split among two separate linkage groups (chromosomes) in M. domestica. Comparative analysis with eight other vertebrate species suggests strong conservation of the BMP6-PRL synteny among birds and mammals, although the BMP6-PRL-MHC-ME1 synteny is not conserved.     

Genomic maps of some strains within the Mycoplasma mycoides cluster

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.     

SINEs, evolution and genome structure in the opossum

Short INterspersed Elements (SINEs) are non-autonomous retrotransposons, usually between 100 and 500 base pairs (bp) in length, which are ubiquitous components of eukaryotic genomes. Their activity, distribution, and evolution can be highly informative on genomic structure and evolutionary processes. To determine recent activity, we amplified more than one hundred SINE1 loci in a panel of 43 M. domestica individuals derived from five diverse geographic locations. The SINE1 family has expanded recently enough that many loci were polymorphic, and the SINE1 insertion-based genetic distances among populations reflected geographic distance. Genome-wide comparisons of SINE1 densities and GC content revealed that high SINE1 density is associated with high GC content in a few long and many short spans. Young SINE1s, whether fixed or polymorphic, showed an unbiased GC content preference for insertion, indicating that the GC preference accumulates over long time periods, possibly in periodic bursts. SINE1 evolution is thus broadly similar to human Alu evolution, although it has an independent origin. High GC content adjacent to SINE1s is strongly correlated with bias towards higher AT to GC substitutions and lower GC to AT substitutions. This is consistent with biased gene conversion, and also indicates that like chickens, but unlike eutherian mammals, GC content heterogeneity (isochore structure) is reinforced by substitution processes in the M. domestica genome. Nevertheless, both high and low GC content regions are apparently headed towards lower GC content equilibria, possibly due to a relative shift to lower recombination rates in the recent Monodelphis ancestral lineage. Like eutherians, metatherian (marsupial) mammals have evolved high CpG substitution rates, but this is apparently a convergence in process rather than a shared ancestral state.     

Mapping of Defense Response Gene Homologs and Their Association with Resistance Loci in Maize

Defense response genes in higher plant species are involved in a variety of signal transduction pathways and biochemical reactions to counterattack invading pathogens. In this study, a total of 366 non-redundant defense response gene homologs （DRHs）, including 124 unigenes/expressed sequence tags, 226 tentative consensuses, and 16 DRH contigs have been identified by mining the Maize Genetics and Genomics and The Institute for Genomic Research maize databases using 35 essential defense response genes. Of 366 DRHs, 202 are mapped to 152 loci across ten maize chromosomes via both the genetic and in silico mapping approaches. The mapped DRHs seem to cluster together rather than be evenly distributed along the maize genome. Approximately half of these DHRs are located in regions harboring either major resistance genes or quantitative trait loci （QTL）. Therefore, this comprehensive DRH linkage map will provide reference sequences to identify either positional candidate genes for resistance genes and/or QTLs or to develop makers for fine-mapping and marker-assisted selection of resistance genes and/or QTLs.     

Gene mapping in marsupials: Detection of an ancient autosomal gene cluster

The genes HRAS, HBB, and CAT, which are located together on the short arm of human chromosome 11, appear to be part of a conserved synteny group found in many eutherian mammals. These genes were mapped to the chromosomes of two marsupial (metatherian) species by in situ hybridization. All three genes were located together on chromosome 3 in Macropus eugenii. Only HRAS and CAT were used to probe Dasykaluta rosamondae metaphases and these genes both mapped to chromosome 4. This suggests that the HRAS-HBB-CAT gene cluster has been conserved at least since the metatherians and eutherians diverged some 130 million years ago. These findings support the concept of a mammalian genome that has remained highly conserved throughout evolution.