Susceptibility of muridae cell lines to ecotropic murine leukemia virus and the cationic amino acid transporter 1 viral receptor sequences: implications for evolution of the viral receptor.

Ecotropic murine leukemia viruses (Eco-MLVs) infect mouse and rat, but not other mammalian cells, and gain access for infection through binding the cationic amino acid transporter 1 (CAT1). Glycosylation of the rat and hamster CAT1s inhibits Eco-MLV infection, and treatment of rat and hamster cells with a glycosylation inhibitor, tunicamycin, enhances Eco-MLV infection. Although the mouse CAT1 is also glycosylated, it does not inhibit Eco-MLV infection. Comparison of amino acid sequences between the rat and mouse CAT1s shows amino acid insertions in the rat protein near the Eco-MLV-binding motif. In addition to the insertion present in the rat CAT1, the hamster CAT1 has additional amino acid insertions. In contrast, tunicamycin treatment of mink and human cells does not elevate the infection, because their CAT1s do not have the Eco-MLV-binding motif. To define the evolutionary pathway of the Eco-MLV receptor, we analyzed CAT1 sequences and susceptibility to Eco-MLV infection of other several murinae animals, including the southern vole (Microtus rossiaemeridionalis), large Japanese field mouse (Apodemus speciosus), and Eurasian harvest mouse (Micromys minutus). Eco-MLV infection was enhanced by tunicamycin in these cells, and their CAT1 sequences have the insertions like the hamster CAT1. Phylogenetic analysis of mammalian CAT1s suggested that the ancestral CAT1 does not have the Eco-MLV-binding motif, like the human CAT1, and the mouse CAT1 is thought to be generated by the amino acid deletions in the third extracellular loop of CAT1.

Postcopulatory sexual selection increases ATP content in rodent spermatozoa.

Sperm competition often leads to increase in sperm numbers and sperm quality, and its effects on sperm function are now beginning to emerge. Rapid swimming speeds are crucial for mammalian spermatozoa, because they need to overcome physical barriers in the female tract, reach the ovum, and generate force to penetrate its vestments. Faster velocities associate with high sperm competition levels in many taxa and may be due to increases in sperm dimensions, but they may also relate to higher adenosine triphosphate (ATP) content. We examined if variation in sperm ATP levels relates to both sperm competition and sperm swimming speed in rodents. We found that sperm competition associates with variations in sperm ATP content and sperm-size adjusted ATP concentrations, which suggests proportionally higher ATP content in response to sperm competition. Moreover, both measures were associated with sperm swimming velocities. Our findings thus support the idea that sperm competition may select for higher ATP content leading to faster sperm swimming velocity.

Evolution of the genomic recombination rate in murid rodents.

Although very closely related species can differ in their fine-scale patterns of recombination hotspots, variation in the average genomic recombination rate among recently diverged taxa has rarely been surveyed. We measured recombination rates in eight species that collectively represent several temporal scales of divergence within a single rodent family, Muridae. We used a cytological approach that enables in situ visualization of crossovers at meiosis to quantify recombination rates in multiple males from each rodent group. We uncovered large differences in genomic recombination rate between rodent species, which were independent of karyotypic variation. The divergence in genomic recombination rate that we document is not proportional to DNA sequence divergence, suggesting that recombination has evolved at variable rates along the murid phylogeny. Additionally, we document significant variation in genomic recombination rate both within and between subspecies of house mice. Recombination rates estimated in F(1) hybrids reveal evidence for sex-linked loci contributing to the evolution of recombination in house mice. Our results provide one of the first detailed portraits of genomic-scale recombination rate variation within a single mammalian family and demonstrate that the low recombination rates in laboratory mice and rats reflect a more general reduction in recombination rate across murid rodents.

Modulation of Fgf3 dosage in mouse and men mirrors evolution of mammalian dentition.

A central challenge in evolutionary biology is understanding how genetic mutations underlie morphological changes. Because highly calcified enamel enables preservation of detailed dental features, studying tooth morphology enables this question to be addressed in both extinct and extant species. Previous studies have found that mutant mice can have severe abnormalities in tooth morphology, and several authors have explored the evolutionary implications of tooth number modifications in mutants. However, although they can potentially shed much light on evolutionary mechanisms, anomalies in tooth shape remain poorly studied. Here, we report that alterations in dosage of the Fgf3 gene cause morphological changes in both genetically engineered mutant mice and in human patients. By comparing the dental morphologies in mice and humans carrying Fgf3 mutations with primitive rodent and primate fossils, we determined that decreases in dosage of Fgf3 lead to phenotypes that resemble the progressive reappearance of ancestral morphologies. We propose that modifications in the FGF signaling pathway have played an important role in evolution of mammalian dentition by giving rise to new cusps and interconnecting cusps by new crests. We anticipate that our multidisciplinary study will advance the detailed correlation of subtle dental modifications with genetic mutations in a variety of mammalian lineages.

Hidden heterochromatin: characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.

Cell-cycle protein modifications in the kidney of Mus spretus from Doñana National Park.

On April 1998 a tailing dam of the Aznalcollar pyrite mine partially collapsed and released to the Guadiamar river acidic water and mud containing toxic metals threatening the Doñana National Park, a Spanish wildlife reserve located near the estuary of Guadalquivir river. To assess the possible biological effects on terrestrial ecosystems, biochemical markers were assayed in the kidneys of Algerian mice (Mus spretus) collected in several areas of Doñana and Guadiamar river. Biomarkers assayed are proteins involved in cell cycle regulation, in particular cyclins and their associated kinases, and some cell cycle inhibitors. Moreover Mitogen Activated Protein Kinases (MAPK), a signal transduction system involved in cell division, p53, a protein involved in growth arrest after DNA damage, and HSP70, an early stress-induced protein, were assayed. The kidneys of animals collected one year after the ecological disaster had increased levels of PCNA (proliferating cell nuclear antigen), indicating an increased number of cells in the S phase of cell cycle. This shift of cells from G0 to S phase is due to increased levels of cyclins D1, E, and A, to decreased levels of p21 and p27 cdk inhibitors, and to activation of MAPK cascade. On the other hand, p53 and HSP70 levels are not changed. These data demonstrate that the presence of toxic metals after ecological disaster provoked the induction of kidney cell proliferation interpretable as a compensatory cell growth after tissue damage and apoptosis, and that could lead to the genomic instability characteristic of cancer cell.

The genetic basis of adaptation: lessons from concealing coloration in pocket mice.

Recent studies on the genetics of adaptive coat-color variation in pocket mice (Chaetodipus intermedius) are reviewed in the context of several on-going debates about the genetics of adaptation. Association mapping with candidate genes was used to identify mutations responsible for melanism in four different populations of C. intermedius. Here, I review four main results (i) a single gene, the melanocortin-1-receptor (Mc1r), appears to be responsible for most of the phenotypic variation in color in one population, the Pinacate site; (ii) four or fewer nucleotide changes at Mc1r appear to be responsible for the difference in receptor function; (iii) studies of migration-selection balance suggest that the selection coefficient associated with the dark Mc1r allele at the Pinacate site is large; and (iv) different (unknown) genes underlie the evolution of melanism on three other lava flows. These findings are discussed in light of the evolution of convergent phenotypes, the average size of phenotypic effects underlying adaptation, the evolution of dominance, and the distinction between adaptations caused by changes in gene dosage versus gene structure.

Differential gene expression in yellow-necked mice Apodemus flavicollis (Rodentia, Mammalia) with and without B chromosomes.

Most B chromosomes are heavily heterochromatic, promoting the general idea that they are genetically inert. The B chromosomes of Apodemus flavicollis are euchromatic and show a high degree of homology with the A chromosomes. The euchromatic nature of B chromosomes in A. flavicollis suggests that they may carry active genes and have transcriptional activity. We applied the differential display reverse transcription-polymerase chain reaction (DD RT-PCR) in order to analyze and compare gene expression in animals possessing B chromosomes and animals without B chromosomes. After a second and third round of amplification, three cDNA fragments were differentially expressed in +B mice compared with 0B animals. These cDNAs were Chaperonin containing TCP-1, subunit 6b (zeta) (CCT6B), Fragile histidine triad gene (FHIT) and hypothetical gene XP transcript. The differential expression pattern was confirmed by Real Time RT-PCR. We suggest that altered expression of these important genes is due to the presence of B chromosomes. In elevating the expression of these genes, B chromosomes of A. flavicollis affect some of the crucial processes in the cell. The significance of these effects and the nature of B chromosomes of A. flavicollis are discussed in the context of the data presented.

Phylogeny and divergence-date estimates of rapid radiations in muroid rodents based on multiple nuclear genes.

The muroid rodents are the largest superfamily of mammals, containing nearly one third of all mammal species. We report on a phylogenetic study comprising 53 genera sequenced for four nuclear genes, GHR, BRCA1, RAG1, and c-myc, totaling up to 6400 nucleotides. Most relationships among the subfamilies are resolved. All four genes yield nearly identical phylogenies, differing only in five key regions, four of which may represent particularly rapid radiations. Support is very strong for a fundamental division of the mole rats of the subfamilies Spalacinae and Rhizomyinae from all other muroids. Among the other "core" muroids, a rapid radiation led to at least four distinct lineages: Asian Calomyscus, an African clade of at least four endemic subfamilies, including the diverse Nesomyinae of Madagascar, a hamster clade with maximum diversity in the New World, and an Old World clade including gerbils and the diverse Old World mice and rats (Murinae). The Deomyinae, recently removed from the Murinae, is well supported as the sister group to the gerbils (Gerbillinae). Four key regions appear to represent rapid radiations and, despite a large amount of sequence data, remain poorly resolved: the base of the "core" muroids, among the five cricetid (hamster) subfamilies, within a large clade of Sigmodontinae endemic to South America, and among major geographic lineages of Old World Murinae. Because of the detailed taxon sampling within the Murinae, we are able to refine the fossil calibration of a rate-smoothed molecular clock and apply this clock to date key events in muroid evolution. We calculate rate differences among the gene regions and relate those differences to relative contribution of each gene to the support for various nodes. The among-gene variance in support is greatest for the shortest branches. We present a revised classification for this largest but most unsettled mammalian superfamily.

Sequence analysis of cDNA encoding follicle-stimulating hormone and luteinizing hormone beta-subunits in the Mastomys (Praomys coucha).

To examine the molecular basis of efficient superovulation in the Mastomys (Praomys coucha), the cDNA sequences of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) beta-subunits were determined and compared with those of other mammals. FSHbeta and LHbeta cDNAs were 1606 and 513 bp long, respectively, from the 5'-ends to the putative polyA sites. The deduced sequences of the FSHbeta and LHbeta precursor proteins were 130 and 141 amino acids in length, respectively. The amino acid sequences of both mastomys hormone subunits showed overall similarity to those of other rodents. In particular, the N-terminus of the FSHbeta precursor protein is of the MM-type, like those of mice and rats, which suggests that the MM-type is characteristic of the subfamily Murinae. As we reported earlier for the Mongolian gerbil, the use of hCG for superovulation of mastomys might need to be re-evaluated, due to the low homology between hCG and rodent LH sequences.

Comparative analysis of micro and macro B chromosomes in the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH.

Comparative analysis of micro B and macro B chromosomes of the Korean field mouse Apodemus peninsulae, collected in populations from Siberia and the Russian Far East, was performed with Giemsa, DAPI, Ag-NOR staining and chromosome painting with whole and partial chromosome probes generated by microdissection and DOP-PCR. DNA composition of micro B chromosomes was different from that of macro B chromosomes. All analyzed micro B chromosomes contained clusters of DNA repeats associated with regions characterized by an uncondensed state in mitosis. Giemsa and DAPI staining did not reveal these regions. Their presence in micro B chromosomes led to their special morphology and underestimation in size. DNA repeat clusters homologous to DNA of micro B chromosome arms were also revealed in telomeric regions of some macro B chromosomes of specimens captured in Siberian regions. Neither active NORs nor clusters of ribosomal DNA were found in the uncondensed regions of micro B chromosomes. Possible evolutionary pathways for the origin of macro and micro B chromosomes are discussed.

Divergent genetic and epigenetic post-zygotic isolation mechanisms in Mus and Peromyscus.

Interspecific hybridization in the rodent genera Peromyscus and Mus results in abnormal placentation. In the Peromyscus interspecies hybrids, abnormal allelic interaction between an X-linked locus and the imprinted paternally expressed Peg3 locus was shown to cause the placental defects. In addition, loss-of-imprinting (LOI) of Peg3 was positively correlated with increased placental size. As in extreme cases this placental dysplasia constitutes a post-zygotic barrier against interspecies hybridization, this finding was the first direct proof that imprinted genes may be important in speciation and thus in evolution. In the Mus interspecies hybrids, a strong role of an X-linked locus in placental dysplasia has also been detected. However, here we show by backcross and allele specific expression analyses that neither LOI of Peg3 nor abnormal interactions between Peg3 and an X-linked locus are involved in generating placental dysplasia in Mus hybrids, although the placental phenotypes observed in the two genera seem to be identical. In contrast to this, another dysgenesis effect common to Peromyscus and Mus hybrids, altered foetal growth, is caused at least in part by the same X-chromosomal regions in both genera. These findings first underline the strong involvement of the X-chromosome in the genetics of speciation. Secondly, they indicate that disruption of epigenetic states, such as LOI, at specific loci may be involved in hybrid dysgenesis effects in one group, but not in another. Thus, we conclude that even in closely related groups divergent molecular mechanisms may be involved in the production of phenotypically similar post-zygotic barriers against hybridization.

Cloning and mRNA expression of guanylin, uroguanylin, and guanylyl cyclase C in the Spinifex hopping mouse, Notomys alexis.

Guanylin and uroguanylin are peptides that activate guanylyl cyclase C (GC-C) receptors in the intestine and kidney, which causes an increase in the excretion of salt and water. The Spinifex hopping mouse, Notomys alexis, is a desert rodent that can survive for extended periods without free access to water and it was hypothesised that to conserve water, the expression of guanylin, uroguanylin, and GC-C would be down-regulated to reduce the excretion of water in urine and faeces. Accordingly, this study examined the expression of guanylin, uroguanylin, and GC-C mRNA in Notomys under normal (access to water) and water-deprived conditions. Initially, guanylin and uroguanylin cDNAs encoding the full open reading frame were cloned and sequenced. A PCR analysis showed guanylin and uroguanylin mRNA expression in the small intestine, caecum, proximal and distal colon, heart, and kidney. In addition, a partial GC-C cDNA was obtained and GC-C mRNA expression was demonstrated in the proximal and distal colon, but not the kidney. Subsequently, a semi-quantitative PCR method showed that water deprivation in Notomys caused a significant increase in guanylin and uroguanylin mRNA expression in the distal colon, and in guanylin and GC-C mRNA expression in the proximal colon. No significant difference in guanylin and uroguanylin mRNA expression was observed in the kidney. The results of this study indicate that there is, in fact, an up-regulation of the colonic guanylin system in Notomys after 7 days of water deprivation.

Genetic variation and activity of mouse Nod2, a susceptibility gene for Crohn's disease.

Genetic variation in human Nod2 has been associated with susceptibility to Crohn's disease. The mouse Nod2 locus is located at chromosome 8 and composed of 12 exons, 11 of which encode the Nod2 protein. Sequence analysis of Nod2 from 45 different strains of Mus musculus and Mus spretus revealed extensive polymorphism involving all exons of Nod2. Of the 140 polymorphic sites identified, 68 were located in the coding region, of which 28 created amino acid substitutions in Nod2. Expression of mouse Nod2 activated NF-kappaB and conferred responsiveness to bacterial components, an activity that was deficient in mutants corresponding to those associated with susceptibility to Crohn's disease. These studies demonstrate a conserved role for Nod2 in the response to bacterial components and suggest that selective evolutionary pressure exerted by pathogens may have contributed to the high level of variability of Nod2 sequences in both humans and mice.

Tests for positive selection on immune and reproductive genes in closely related species of the murine genus mus.

We examine variation among species of Mus in four genes involved in reproduction and the immune response for evidence of positive selection: the sperm recognition gene Zp-3, the testis-determining locus Sry, the testicular cell surface matrix protein Tcp-1, and the immune system protein beta(2) m. We use likelihood ratio tests in the context of a well-supported phylogeny to determine whether models that allow for positively selected sites fit the sequences better than models that assume purifying selection. We then apply a Bayesian approach to identify particular sites in each gene that have a high posterior probability of being under positive selection. We find no evidence of positive selection on the Tcp-1 gene, but for Zp-3, Sry, and beta(2) m, models that allow for positively selected sites fit the sequences better than alternatives. For each of these genes, we identify sites that have a high (> 95%) posterior probability of being positively selected. For Zp-3, two of these sites occur near the sperm-binding region, while one occurs in a region whose functional role remains unstudied but where the pattern of change predicts functional importance. A single site in Sry shows an elevated rate of replacement substitution but occurs in a region of apparently little functional importance; therefore, relaxation of functional constraints may better explain the rapid evolution of this site. Three sites in beta(2) m have a posterior probability > 50% of being under positive selection. While the functional role for two of these sites is unknown, the third is known to influence the ability of MHC class I molecules to present antigens to the immune system; therefore, the elevated rate of replacement substitutions at this site is consistent with selection acting to promote variability in immune system proteins.

Cytochrome b-based phylogeny of the Praomys group (Rodentia, Murinae): a new African radiation?

Complete cytochrome b gene sequences allows, for the first time, establishing a nearly complete phylogeny among the Praomys group sensu lato. The genera Praomys, Mastomys and Stenocephalemys appear paraphyletic. Myomys is polyphyletic and this genus name probably needs to be restricted to its type species, M. verreauxii. The genera Zelotomys and Colomys appear as sister groups. Mastomys pernanus and Malacomys verschureni nest within the Praomys group, but their generic assignation must be further clarified. The genus Heimyscus appears closest to Praomys than to Hylomyscus. The different lineages probably result from an adaptive radiation at the end of the Miocene.

Preferential transcription of a gene for alpha amylase in the carcinoid tumor of African rodent Mastomys natalensis.

The carcinoid tumor in Mastomys natalensis provides a useful animal model of tumorigenesis. We investigated preferentially transcribed genes in this carcinoid tumor by differential hybridization. Fourteen clones corresponding to high levels of transcription were isolated from a cDNA library. Sequencing analysis and a homology search revealed that the clones corresponded to genes for chromogranin and alpha-amylase. High-level transcription of a gene for alpha-amylase gene in Mastomys carcinoid tumor was confirmed by Northern blotting analysis. Furthermore, Western blotting analysis confirmed the expression of alpha-amylase in tumors at the protein level. Immunohistochemical staining revealed alpha-amylase in the cytoplasm of Mastomys carcinoid tumors. Our results demonstrated that an exocrine enzyme 'amylase' could be produced ectopically by a neuroen docrine tumor.

Mouse pituitary adenylate cyclase-activating polypeptide (PACAP): gene, expression and novel splicing.

PACAP is a conserved neuropeptide present in all vertebrates. In the mouse, the PACAP gene and various mRNAs have been isolated. To further characterize the mouse PACAP gene (Adcyap1), we have confirmed its sequence, identified its chromosomal location on distal chromosome 17 and used RT-PCR and 5'RACE in various tissues to identify eight PACAP mRNAs, four of which are novel. Three of these novel transcripts have alternate 5'UTRs, whereas the fourth is altered within the coding region. This is the first report of alternate splicing within the coding region of the PACAP gene. The expression pattern of PACAP in the mouse during embryonic development and adulthood is known. Here, expression of PACAP in the mouse at four postnatal stages in 12 tissues is assessed. We identify continuous expression of PACAP in the brain and eye and stage-specific expression in the gonads and thymus. The complex splicing of PACAP transcripts may regulate the tissue-and stage-specific expression.

Genomic organization of the mouse src gene. Sequencing of src introns revealed a new chromosome 2 microsatellite marker.

Ten introns interrupting the coding sequence of the mouse src protooncogene were sequenced (in total 11260 bp) and their general characteristics compared with the homologous genes in human and chicken. While the study of genome organization of the src gene was performed only in the inbred mouse strain BALB/cHeA (Mus musculus domesticus), one special region in the intron 5 was also sequenced in additional mouse strains (M. musculus musculus and M. spretus), because the discovered CA and GT repeat array differences could serve as a new polymorphic marker in the chromosome No. 2 and help elucidate some evolutionary relations between mouse strains.