Restriction fragment length polymorphism and evolution of the mouse immunoglobulin constant region gamma loci

Genomic DNA from twelve laboratory mouse strains, in addition to 21 wild-derived strains belonging to different taxa (Mus musculus domesticus, Mus musculus musculus, Mus spretus, Mus macedonicus, a and Mus spicilegus) and four mouse strains that are evolutionarily more distant, were analyzed by Southern blot for polymorphism of the Ig heavy chain constant region isotype (Igh-C) and for the distribution of the duplicated Igh-1 (C2) haplotype. Distinct allelic forms of each Igh-C locus could be defined by restriction fragment length polymorphism (RFLP). In laboratory mouse strains RFLP proved to be more sensitive in the detection of Igh-4 (C1) alleles than serological methods. Taq I digestion allowed the definition of two alleles in the Igh-8 (C3) locus, which is absolutely conserved at the protein levels. More extensive RFLP could be found in wild strains belonging to the subgenus Mus and in the evolutionarily more distant Mus species belonging to other subgenera. In previous studies we have shown that the Igh-1 locus is duplicated in M. m. musculus subspecies. We now extend this observations to the wild mouse strains belonging to M. spicilegus and M. macedonicus species and to the evolutionarily more distant wild mouse strain Mus pahari (subgenus coelomys), which is thought to have diverged from domestic mice about 5 million years ago. In addition, we found a similar RFLP pattern in ten of 18 wild mice trapped in India, suggesting that the haplotype containing the two Igh-1-like genes, organized in tandem as distinct isotypes, is widely spread in natural populations. The evolution of murine Igh-C-encoded isotypes is also discussed. Correspondence to: P.-A. Cazenave.     

First Insights into the Phylogenetic Diversity of Mycobacterium tuberculosis in Nepal

Background

Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown.

Methods and Findings

We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status.

Conclusions

We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.     

Chromosomal and molecular divergence in the Indian pygmy field mice Mus booduga-terricolor lineage of the subgenus Mus

Mus booduga and Mus terricolor both have 2n=40. Unlike M. booduga, with all acrocentric chromosomes, M. terricolor invariably has large submetacentric X and acrocentric Y due to an increase of heterochromatin. In contrast to the conservative karyotype of the co-existing sibling species booduga, three chromosome types of terricolor are found in different populations and their divergent karyotypes have autosomal heterochromatin variations established in the homozygous condition. The average genetic distance determined from electrophoretic study of 20 protein loci ranges from lowest (D=0.106) between chromosome types I & II to highest (D=0.185) between types II & III. In terricolor, booduga and M. m. tytleri high mean values of variations per locus (range A=1.604 to 1.928) and heterozygosity per individual per locus (range H=0.180 to 0.336) have been observed. Sequence divergence of 0.39 to 1.2%, calculated from restriction profiles of mtDNA, shows that the terricolor chromosome types have diverged recently. Hybridizations between type I females and type III males gave a preponderance of males in the F1 with varying degrees of sterility. The terricolor complex is an interesting system for critical probing for the role of heterochromatin in the process of speciation. MtDNA, protein loci and AT-rich musculus-related major and minor satellite DNA data indicate that progenitors of the booduga-terricolor lineage might have evolved simultaneously with the caroli-cookii-cervicolor lineage in the evolution of the subgenus Mus.     

Modern Lineages of Mycobacterium tuberculosis Exhibit Lineage-Specific Patterns of Growth and Cytokine Induction in Human Monocyte-Derived Macrophages

Background

Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis.

Methods

Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction.

Results

In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p = 0.0024; lineage 4 vs. lineage 3 p = 0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40.

Conclusions

Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.     

Allelic variants of Ly-5 in inbred and natural populations of mice

Allelic variants of Ly-5 in inbred commensal and other natural populations of mice were analyzed by patterns of restriction fragment length polymorphisms (RFLP) and Southern hybridization using an Ly-5 cDNA probe and by cell-surface staining with a panel of antibodies directed against polymorphic and nonpolymorphic Ly-5 determinants. New Ly-5 alleles were defined by RFLPs generated by both Eco RI and Bam HI restriction enzyme digests. The Mus musculus subspecies and other species within the genus Mus showed a strong correlation between allelic variants defined by restriction enzymes and serologic specificities. The data also suggest the conservation of the Ly-5 gene throughout the genus Mus.     

Isolation,Characterization, and Evolutionary Divergence of Mouse RNase 6: Evidence for Unusual Evolution in Rodents

The evolution of the ribonuclease A (RNase A) vertebrate-specific enzyme family is interesting in that specific gene lineages appear to be responding to unique selective pressures in wildly diverse manners to generate proteins that are capable of reducing the infectivity of viruses, killing systemic pathogens, and inducing the growth of blood vessels all while maintaining the signature motifs of a ribonuclease. In this paper, we present the DNA sequence and gene structure of Mus musculus RNase 6 and examine the expression pattern and enzymatic activity of the recombinant protein. M. musculus RNase 6 has a limited expression pattern compared to human RNase 6 and is an efficient ribonuclease, with a catalytic efficiency 17-fold higher than that of human protein. Evo- lutionary analysis reveals that RNase 6 was subject to unusual evolutionary forces (d_N/d_S=1.2) in an ancestral rodent lineage before the separation of Mus and Rattus. However, more recent evolution of rodent RNase 6 has been relatively conserved, with an average d_N/d_S of 0.66. These data suggest that the ancestral rodent RNase 6 was subject to accelerated evolution, resulting in the conserved modern gene, which most likely plays an important role in mouse physiology.Reviewing Editor: Dr. Lauren Ancel MeyersThe GenBank accession numbers for the new genes presented here are as follows: Mus musculus, AY545655; Rattus norvegicus, AY545654; Mus spicilegus, AY545653; Mus caroli, AY545651; and Mus pahari, AY545652.     

Cloning and characterization of class I Mhc genes of the zebrafish,Brachydanio rerio

The zebrafish (Brachydanio rerio) offers many advantages for immunological and immunogenetic research and has the potential for becoming one of the most important nonmammalian vertebrate research models. With this in mind, we initiated a systematic study of the zebrafish major histocompatibility complex (Mhc) genes. In this report, we describe the cloning and characteristics of the zebrafish class I A genes coding for the chains of the heterodimer and thus complete the identification of all four classes and subclasses of the Mhc in this species. We describe the full class I cDNA sequence as well as the exon-intron organization of the class I A genes, including intron sequences. We identify three families of class I A genes which we designate Bree-UAA,-UBA, and -UCA. The three families originated about the time of the divergence of cyprinid and salmonid fishes. All three families are members of an ancient lineage that diverged from another, older lineage also represented in cyprinid fishes before the radiation of teleost orders. The fish class I A genes therefore evolve differently from mammalian class I A genes, in which the establishment of lineages and families mostly postdates the divergence of orders.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers Z46776–Z46779     

Evolution of murine α1-proteinase inhibitors: Gene amplification and reactive center divergence

The organization and sequence of genes encoding the ₁-proteinase inhibitor (₁PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of ₁PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian ₁PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10–13 million years ago. Thus, ₁PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the ₁PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of ₁PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.Correspondence to: F.G. Berger     

Multiple sites of crossing over within the Eb recombinational hotspot in the mouse

The Eb gene of the mouse major histocompatibility complex (MHC) contains a well-documented hotspot of recombination. Twelve cases of intra-Eb recombination derived from the b, d, k and s alleles of the Eb gene were sequenced to more precisely position the sites of meiotic recombination. This analysis was based on positioning recombination breakpoints between nucleotide polymorphisms found in the sequences of parental haplotypes. All twelve cases of recombination mapped within the second intron of the Eb gene. Six of these recombinants, involving the k and s haplotypes, mapped to two adjoining DNA segments of 394 and 955 base pairs (bp) in the 3 half of the intron. In an additional two cases derived by crossing over between the d and s alleles, breakpoints were positioned to adjoining segments of 28 and 433 bp, also in the 3 half of the intron. Finally, four b versus k recombinants were mapped to non-contiguous segments of DNA covering 2.9 kb and 1005 bp of the intron. An analysis of the map positions of crossover breakpoints defined in this study suggests that the second intron of the Eb gene contains a recombinational hotspot of approximately 800–1000 bp which contains at least two closely linked recombinationally active sites or segments. Further examination of the sequence data also suggests that the postulated location for the recombinational hotspot corresponds almost precisely to an 812 bp sequence that shows nucleotide sequence similarity to the MT family of middle repetitive DNA.     

On the trail of Neolithic mice and men towards Transcaucasia: zooarchaeological clues from Nakhchivan (Azerbaijan)

Transcaucasia comprises a key region for understanding the history of both the hybrid zone between house mouse lineages and the dispersal of the Neolithic way of life outside its Near Eastern cradle. The opportunity to document the colonization history of both men and mice in Transcaucasia was made possible by the discovery of mouse remains accumulated in pits from a 6000‐year‐old farming village in the Nakhchivan (Autonomous Republic of Nakhchivan, Azerbaijan). The present study investigated their taxonomy and most likely dispersal path through the identification of the Mus lineage to which they might belong using a geometric morphometric approach of dental traits distances between archaeological and modern populations of the different Mus lineages of South‐West Asia. We demonstrate that the mouse remains trapped in the deep storage pits of the dwelling belong to the Mus musculus domesticus from the Near East, with dental shapes similar to current populations in Northern Syria. These results strongly suggest that the domesticus lineage was dispersed into Transcaucasia from the upper Euphrates valley by Neolithic migration, some time between the 7th and 5th millennium BC, providing substantial evidence to back up the scenario featuring near‐eastern stimuli in the emergence of agriculture in the South Caucasus. The domesticus mitochondrial DNA signature of the current house mouse in the same location 5000 years later, as well as their turnover towards a subspecies musculus/castaneus phenotype, suggests that early domesticus colonizers hybridized with a later musculus (and maybe castaneus) dispersal originating from south of the Caspian Sea and/or Northern Caucasia. © 2013 The Linnean Society of London     

Changes in Mycobacterium tuberculosis Genotype Families Over 20 Years in a Population-Based Study in Northern Malawi

Background

Despite increasing interest in possible differences in virulence and transmissibility between different genotypes of M. tuberculosis, very little is known about how genotypes within a population change over decades, or about relationships to HIV infection.

Methods and Principal Findings

In a population-based study in rural Malawi we have examined smears and cultures from tuberculosis patients over a 20-year period using spoligotyping. Isolates were grouped into spoligotype families and lineages following previously published criteria. Time trends, HIV status, drug resistance and outcome were examined by spoligotype family and lineage. In addition, transmissibility was examined among pairs of cases with known epidemiological contact by assessing the proportion of transmissions confirmed for each lineage, on the basis of IS6110 RFLP similarity of the M tuberculosis strains. 760 spoligotypes were obtained from smears from 518 patients from 1986–2002, and 377 spoligotypes from cultures from 347 patients from 2005–2008. There was good consistency in patients with multiple specimens. Among 781 patients with first episode tuberculosis, the majority (76%) had Lineage 4 (“European/American”) strains; 9% had Lineage 3 (“East-African/Indian”); 8% Lineage 1 (“Indo-Oceanic”); and 2% Lineage 2 (“East-Asian”); others unclassifiable. Over time the proportion of Lineage 4 decreased from >90% to 60%, with an increase in the other 3 lineages (p<0.001). Lineage 1 strains were more common in those with HIV infection, even after adjusting for age, sex and year. There were no associations with drug resistance or outcome, and no differences by lineage in the proportion of pairs in which transmission was confirmed.

Conclusions

This is the first study to describe long term trends in the four M. tuberculosis lineages in a population. Lineage 4 has probably been longstanding in this population, with relatively recent introductions and spread of Lineages1–3, perhaps influenced by the HIV epidemic.     

Structural and genetic properties of the Eb recombinational hotspot in the mouse

The Eb gene of the mouse contains a recombinational hotspot which plays a predominant role in meiotic crossing-over within the I region of the mouse major histocompatibility complex (MHC). The nucleotide sequences of five recombinants derived from H-2 ^k /H-2 ^b heterozygotes at the Eb locus placed the sites of recombination in each recombinant haplotype within a 2.9 kilobase (kb) segment located fully within the second intron of the Eb gene. Further resolution of the crossover sites was not possible since the nucleotide sequences of the parental and recombinant haplotypes are identical within this segment. The molecular characterization of these five recombinants considered in conjunction with three previously reported intra-Eb recombinants indicates that there are at least two distinct sites of recombination within the Eb recombinational hotspot. In a related study, an examination of the nucleotide sequence of the H-2 ^p allele of the Eb gene revealed a major genetic rearrangement in the 5' half of the intron in this haplotype. A 597 base pair (bp) nucleotide sequence found in the H-2 ^p haplotype is replaced by a 1634 bp segment found in the H-2 ^b and H-2 ^k haplotypes. Sequence analysis of this 1634 bp segment shows strong nucleotide sequence similarity to retroposon long terminal repeat (LTR), env, and pol genes indicating that this segment of the second intron has evolved through retroposon insertion. The location of these retroposon sequences within the 2.9 kb recombination segment defined by the five H-2 ^k /H-2 ^b recombinant haplotypes suggests a possible relationship between these retroviral elements and site-specific recombination within the second intron of the Eb gene. Offprint requests to: H. C. Passmore     

Mhc class II B gene evolution in East African cichlid fishes

 A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the class II B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization. Received: 6 December 1999 / Revised: 15 February 2000     

Genetics and evolution of the acute phase proteins in mice

Summary In mammals, a number of liver-derived plasma proteins, termed acute phase reactants, are induced during an inflammation response. We have studied genetic variation in the structure and expression of several of these proteins in a variety of inbred and wild-derived mice. In a genetic cross, electrophoretic polymorphisms for the two ₁-acid glycoproteins, AGP-1 and AGP-2, co-segregated in 58 backcross progeny, indicating that either a single gene or two tightly-linked genes on chromosome 4 encode the AGPs. In the same backcross, segregation of variation in haptoglobin structure showed that the gene encoding this acute phase reactant is on chromosome 8. Structural variation in serum amyloid A correlated with restriction fragment length polymorphisms in the Saa gene determined by Taylor and Rowe (1984). Analysis of a number of highly diverged species of mice indicated that AGP expression has undergone considerable modification during evolution of the Mus genus; this is associated with alterations in Agp gene organization, which may include species-specific amplification and/or deletion events.     

Demographic and phylogeographic histories of two venomous North American snakes of the genus Agkistrodon

Many studies have revealed that lineages currently inhabiting formerly glaciated areas were pushed into southern glacial refugia and have expanded into their modern range since the last glacial maximum. There have been few studies that compare the effects of glacial cycles on lineage diversification and historical demography in closely related species with overlapping ranges. In this study we compare phylogeographic structure, historical demography, and approximate lineage age in two closely related and broadly co-occurring venomous snakes in eastern North America, the cottonmouth (Agkistrodon piscivorus) and copperhead (A. contortrix) using sequences from the mtDNA gene cytochrome b. We inferred three geographic lineages of A. contortrix and two of A. piscivorus with no common geographic or temporal pattern of lineage diversification identified for these species. Lineage diversification occurred in the Late Pliocene for A. piscivorus (2.5 mya) and in the Early Pleistocene for A. contortrix (1.5 mya). Demographic estimates revealed population expansion following the last glacial maximum (20,000 years ago) in two lineages of A. contortrix (the Central clade and Eastern clade) and one lineage of A. piscivorus (the Continental clade). The Florida clade of A. piscivorus is the only lineage for which constant population size through time was inferred, possibly due to stable populations persisting in areas unaffected by glacial advances. Our data suggest that unique habitat preferences may have shaped both the phylogeographic and demographic histories of each species.     

The Presence/Absence Polymorphism and Evolution of the p53 Pseudogene in the GenusMus

Distribution of the p53 pseudogene within the house mouse species (genusMus) was studied with polymerase chain reaction for 37 individuals that were caught at different localities. Pseudogene-specific fragments were detected in some, but not all, individuals ofMus musculussubspecies regardless of locality and type of subspecies. In addition, 3 of 7 individuals belonging to differentMusspecies carried the pseudogene in their genomes. These results show the existence of an interspecific presence/absence polymorphism of the p53 pseudogene in mice. Sequence analysis of 11 amplified 0.3-kb fragments suggested that the pseudogene originated in an ancestral mouse about 7 million years ago. Thus alleles with and without the p53 pseudogene have persisted through the mice speciation. The evolutionary rate for the p53 functional gene was also estimated to be about 3.3 × 10⁻⁹per nucleotide site per year.     

Evolution of introns and exons of class II major histocompatibility complex genes of vertebrates

 The phylogenetic relationships and patterns of nucleotide substitution were compared for introns and exons of class II major histocompatibility complex (MHC) genes in three datasets: human DRB1, human DQA1, and cyprinid fish DAB1. In both human DRB1 and cyprinid DAB1, there was strong evidence that recombination events between alleles have occurred in such a way that intron and exon sequences of a given allele do not necessarily share the same evolutionary history. In the case of human DRB1, recombination was found to have homogenized intron 1 and intron 2 sequences relative to exon 2 sequences within lineages of alleles but not between lineages. As a result, mean divergence times of intron sequences are much more recent than those of exonic sequences. Thus, the divergence time of DRB1 introns cannot be used to date that of exons in the same alleles, and the hypothesis that most human DRB1 polymorphism is of very recent origin is not supported. Received: 5 September 1999 / Revised: 30 December 1999     

Morphological and genetic diversity of the thermophilic cyanobacterium,Mastigocladus laminosus (Stigonematales,Cyanobacteria) from Japan and Myanmar

The morphology and phylogeny of 13 strains of a thermophilic cyanobacterium, Mastigocladus laminosus Cohn, isolated from hot springs in Japan and Myanmar were analyzed to determine taxonomy and biogeography. From the morphological observations of cell size, there were significant differences among strains. Phylogenetic analyses based on 16S rRNA gene sequences revealed two lineages: Lineages I and II. Lineage I consisted of strains collected in Japan and reference strains from a previous study (CCMEE 5329 and 5331, Hakone, Japan); Lineage II included all of the Myanmar strains and one Japanese strain, and was a novel lineage in phylogeographic studies on M. laminosus. Since strains in the Lineage II tended to have larger cells than those in the Lineage I, the morphological and phylogenetic lineages corresponded well with each other.