Rat kappa-chain allotypes. III. Serological and genetic analysis of RI-1b cross-reactivity in Australian Rattus

The presence of rat kappa-chain allotype specificities (RI-1a and b) has been studied in 13 subspecies of the seven native Australian species of Rattus. RI-1a reactivity was not detected among these rats. On the other hand, extensive cross-reactivity was seen with RI-1b, some sera cross-reacting totally (R. leucopus cooktownensis), some not at all (R. colletti), and the remainder showing at least two distinct levels of partial cross-reactivity, confirming the existence of multiple specificities for RI-1b. Three subspecies show polymorphism with respect to RI-1b cross-reactivity (R. sordidus, R. colletti, and R. l. leucopus) and in one case (leucopus) breeding studies have indicated that there is allelic inheritance of this trait. The segregation of RI-1b reactivity has been studied in crosses and backcrosses made between species differing in their RI-1b reactivity, and the results are consistent with the existence of codominant alleles at a single locus. The fact that these species differ extensively in their karyotype opens the door to possible chromosomal localization of this and other genetic traits.     

Polymorphism and mapping of the class II genes in the rat: RT1.B,RT1.D,and RT1.H,a new DP-like region

The major histocompatibility complex of the rat (RTI) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the - and -chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has - and -chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1. B. The RT1. H and RT1.H genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H and D genes have high levels of polymorphism; B , B ,and H have intermediate levels of polymorphism, and D has a low level of polymorphism.     

Biochemical differentiation among karyotypic forms of Australian Rattus

Isozyme electrophoresis of up to 55 loci, and microcomplement fixation of albumin were used to assess the extent of structural gene divergence among karyotypic forms of Australian Rattus. The results show that the Australian Rattus are monophyletic with respect to R. rattus or R. norvegicus. Within the Australian Rattus, rates of chromosomal evolution have varied enormously, the highest rates being found among members of the R. sordidus group, where extensive chromosomal repatterning has occurred with little or no structural gene divergence.     

Mitochondrial iso-citrate dehydrogenase variation in the Australian bush-rat, Rattus fuscipes greyii

Four reproductively isolated populations of the Australian bush-rat, Rattus fuscipes greyii , are polymorphic for electrophoretic variants of the mitochondrial enzyme, NADP-dependent iso-citrate dehydrogenase (M-Idh). The electrophoretic patterns of M-Idh and a small amount of breeding data are in agreement with the hypothesis that the variation is controlled by two alleles at an autosomal locus, Idh-2. In three relatively large populations, the proportion of heterozygotes at the Idh-2 locus ranges from 0.07 to 0.23, while on a small off-shore island it is 0.60. In this latter population there is an excess of heterozygotes which is on the borderline of statistical significance, suggesting that heterotic selection may be maintaining the polymorphism. Populations on eight other small islands are monomorphic for the allele which is the most frequent in the large populations. In most species M-Idh is less variable than most other enzymic proteins and the results presented here constitute one of the few published examples of an extensive M-Idh polymorphism.     

Evolutionary adaptation to environmental pH in experimental lineages of Escherichia coli

This study uses the enteric bacterium Escherichia coli as an experimental system to examine evolutionary responses of bacteria to an environmental acidic-alkaline range between pH 5.3 and 7.8 (15-5000 nM [H(+)]). Our goal was both to test general hypotheses about adaptation to abiotic variables and to provide insights into how coliform organisms might respond to changing conditions inside and outside of hosts. Six replicate lines of E. coli evolved for 2000 generations at one of four different constant pH conditions: pH 5.3, 6.3, 7.0, or 7.8. Direct adaptation to the evolutionary environment, as well as correlated changes in other environments, was measured as a change in fitness relative to the ancestor in direct competition experiments. The pH 5.3 group had the highest fitness gains, with a highly significant increase of 20%. The pH 7.8 group had far less significant gains and much higher variance among its lines. Analysis of individual lines within these two groups revealed complex patterns of adaptation: all of the pH 5.3 lines exhibited trade-offs (reduced fitness in another environment), but only 33% of the pH 7.8 lines showed such trade-offs and one of the pH 7.8 lines demonstrated exaptation by improving fitness in the pH 5.3 environment. Although there was also prevalent exaptation in other groups to the acidic environment, there were no such cases of exaptation to alkalinity. Comparison across the entire experimental pH range revealed that the most acidic lines, the pH 5.3 group, were all specialists, in contrast to the pH 6.3 lines, which were almost all generalists. That is, although none of the pH 5.3 lines showed any correlated fitness gains, all of the pH 6.3 lines did.     

Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis

Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.     

Independent integration of rodent identifier (ID) elements into orthologous sites of some RT6 alleles of Rattus norvegicus and Rattus rattus

The two alleles of the rat T-cell differentiation alloantigen RT6 are highly divergent and their expression is distinctively regulated. While the majority of T cells of RT6a/RT6b heterozygous laboratory rats (Rattus norvegicus) expresses both alleles, a subpopulation expresses only RT6b. To identify cis-regulatory elements that potentially control monoallelic expression, we compared the sequences of both alleles. A striking difference is the presence or absence of a rodent identifier (ID) sequence in intron 7. All investigated inbred RT6a rat strains (n=7) had this integration, while it was absent in all investigated RT6b rats (n=9). An ID element was also identified at precisely the same integration site in one RT6 allele of the closely related species Rattus rattus. The ID elements of both species showed nucleotide substitutions characteristic of different subfamilies, and their flanking repeats differed in length, indicating that two independent integration events had occurred into the same site adjacent to a mammalianwide interspersed repeat. Analysis of the surrounding sequences did not disclose any motifs to explain preferential integration into one allele. Our data indicate that the RT6 alleles diverged about 1 myr ago and that the ID element integrated into the RT6a locus soon after this. We have previously shown that DNA methylation plays an important role in regulating monoallelic RT6 expression. The possibility that the ID element in the RT6a allele interferes with the required demethylation process and thus accounts for monoallelic expression is discussed.     

Open reading frame sequencing and structure-based alignment of polypeptides encoded by RT1-Bb, RT1-Ba, RT1-Db, and RT1-Da alleles

MHC class II genes are major genetic components in rats developing autoimmunity. The majority of rat MHC class II sequencing has focused on exon 2, which forms the first external domain. Sequence of the complete open reading frame for rat MHC class II haplotypes and structure-based alignment is lacking. Herein, the complete open reading frame for RT1-B, RT1-B, RT1-D, and RT1-D was sequenced from ten different rat strains, covering eight serological haplotypes, namely a, b, c, d, k, l, n, and u. Each serological haplotype was unique at the nucleotide level of the sequenced RT1-B/D region. Within individual genes, the number of alleles identified was seven, seven, six, and three and the degree of amino-acid polymorphism between allotypes for each gene was 22%, 16%, 19%, and 0.4% for RT1-B, RT1-B, RT1-D, and RT1-D, respectively. The extent and distribution of amino-acid polymorphism was comparable with mouse and human MHC class II. Structure-based alignment identified the 65–66 deletion, the 84a insertion, the 9a insertion, and the 1a–1c insertion in RT1-B previously described for H2-A. Rat allele-specific deletions were found at RT1-B76 and RT1-D90–92. The mature RT1-D polypeptide was one amino acid longer than HLA-DRB1 due to the position of the predicted signal peptide cleavage site. These data are important to a comprehensive understanding of MHC class II structure-function and for mechanistic studies of rat models of autoimmunity.Nucleotide sequence data reported are available in the GenBank database under the accession numbers AY626180–AY626189 for all RT1-Bb sequences, AY626190–AY626199 for all RT1-Ba sequences, AY626200-AY626209 for all RT1-Db sequences and AY626210–AY626219 for all RT1-Da sequences.     

Evolutionary origins of retroposon lineages of Mhc class II Ab alleles

Major histocompatibility complex (Mhc) class II Ab genes have evolved into three distinct lineages. While lineage 2 alleles differ from lineage 1 alleles by the insertion of a retroposon in intron 2, the basis for the extremely large intron 2 in lineage 3 alleles has heretofore been undetermined. In this report, we demonstrate by nucleotide sequencing that the genomic sequences of prototypic alleles from all three lineages diverge significantly and that lineage 3 is derived from lineage 2 by two insertional events in intron 2. One insert, composed of a member of B1 short interspersed repetitive elements (SINEs), occurs 508 base pairs (bp) 3 of exon 2, and the other, 1141 bp 3 of exon 2 within the retroposon that distinguishes lineage 2 from lineage 1. To assess the evolutionary stability of these lineages and the extent of ancestral polymorphisms of Ab within Mus species, we extended our restriction site polymorphism analysis to include 86 alleles from 120 independently derived H2 haplotypes from 12 separate species and subspecies of Mus. A phylogenetic tree revealing the relationships of these Ab alleles with respect to restriction site polymorphisms, but excluding the retroposon insertions, demonstrated that these lineages have distinctive genomic structures beyond the retroposon polymorphisms. In summary, these mouse Ab genes were produced from successive retroposon insertion events. Lineage 1 and 2 were detected in a variety of Mus species, including Mus caroli, indicating that these lineages diverged more than 2 million years ago. Lineage 3 alleles were found only in the Mus musculus subspecies, suggesting that it diverged from lineage 2 more recently. These results indicate that all three lineages of Ab have persisted through several speciation events in the genus Mus.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60562     

An immunochemical procedure for the purification of rat class I antigens (RT1.A/RT1.E) from detergent-solubilized erythrocytes

An immunochemical procedure using two immunoaffinity columns for the isolation of rat Class I antigens from detergent-solubilized DA erythrocytes is described. The protocol yields a pure Class I preparation as assessed by silver staining of sodium dodecyl sulfate-polyacrylamide gels and comparison of the amino acid composition with that previously determined for H-2K/D. The antigen preparation is capable of specifically blocking the hemagglutination of DA erythrocytes by Class I-specific alloantiserum.     

Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the tertiary

Tertiary macrofossils of the flowering plant family Leguminosae (legumes) were used as time constraints to estimate ages of the earliest branching clades identified in separate plastid matK and rbcL gene phylogenies. Penalized likelihood rate smoothing was performed on sets of Bayesian likelihood trees generated with the AIC-selected GTR+ Gamma +I substitution model. Unequivocal legume fossils dating from the Recent continuously back to about 56 million years ago were used to fix the family stem clade at 60 million years (Ma), and at 1-Ma intervals back to 70 Ma. Specific fossils that showed distinctive combinations of apomorphic traits were used to constrain the minimum age of 12 specific internal nodes. These constraints were placed on stem rather than respective crown clades in order to bias for younger age estimates. Regardless, the mean age of the legume crown clade differs by only 1.0 to 2.5 Ma from the fixed age of the legume stem clade. Additionally, the oldest caesalpinioid, mimosoid, and papilionoid crown clades show approximately the same age range of 39 to 59 Ma. These findings all point to a rapid family-wide diversification, and predict few if any legume fossils prior to the Cenozoic. The range of the matK substitution rate, 2.1-24.6 x 10(-10) substitutions per site per year, is higher than that of rbcL, 1.6- 8.6 x 10(-10), and is accompanied by more uniform rate variation among codon positions. The matK and rbcL substitution rates are highly correlated across the legume family. For example, both loci have the slowest substitution rates among the mimosoids and the fastest rates among the millettioid legumes. This explains why groups such as the millettioids are amenable to species-level phylogenetic analysis with these loci, whereas other legume groups are not.