Unexpected evolutionary diversity in a recently extinct Caribbean mammal radiation

Identifying general patterns of colonization and radiation in island faunas is often hindered by past human-caused extinctions. The insular Caribbean is one of the only complex oceanic-type island systems colonized by land mammals, but has witnessed the globally highest level of mammalian extinction during the Holocene. Using ancient DNA analysis, we reconstruct the evolutionary history of one of the Caribbean''s now-extinct major mammal groups, the insular radiation of oryzomyine rice rats. Despite the significant problems of recovering DNA from prehistoric tropical archaeological material, it was possible to identify two discrete Late Miocene colonizations of the main Lesser Antillean island chain from mainland South America by oryzomyine lineages that were only distantly related. A high level of phylogenetic diversification was observed within oryzomyines across the Lesser Antilles, even between allopatric populations on the same island bank. The timing of oryzomyine colonization is closely similar to the age of several other Caribbean vertebrate taxa, suggesting that geomorphological conditions during the Late Miocene facilitated broadly simultaneous overwater waif dispersal of many South American lineages to the Lesser Antilles. These data provide an important baseline by which to further develop the Caribbean as a unique workshop for studying island evolution.     

Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.     

The complete map of the Ig heavy chain constant gene region reveals evidence for seven IgG isotypes and for IgD in the horse

This report contains the first map of the complete Ig H chain constant (IGHC) gene region of the horse (Equus caballus), represented by 34 overlapping clones from a new bacterial artificial chromosome library. The different bacterial artificial chromosome inserts containing IGHC genes were identified and arranged by hybridization using overgo probes specific for individual equine IGHC genes. The analysis of these IGHC clones identified two previously undetected IGHC genes of the horse. The newly found IGHG7 gene, which has a high homology to the equine IGHG4 gene, is located between the IGHG3 and IGHG4 genes. The high degree of conservation shared between the nucleotide sequences of the IGHG7 and IGHG4 genes is unusual for the IGHG genes of the horse and suggests that these two genes duplicated most recently during evolution of the equine IGHG genes. Second, we present the genomic nucleotide sequence of the equine IGHD gene, which is located downstream of the IGHM gene. Both the IGHG7 and IGHD genes were found to be expressed at the mRNA level. The order of the 11 IGHC genes in the IGH-locus of the horse was determined to be 5'-M-D-G1-G2-G3-G7-G4-G6-G5-E-A-3', confirming previous studies using lambda phage clones, with the exception that the IGHG5 gene was found to be the most downstream-located IGHG gene. Fluorescence in situ hybridization was used to localize the IGHC region to Equus caballus (ECA) 24qter, the horse chromosome corresponding to human chromosome 14, where the human IGH locus is found.     

A new species of recently extinct rice rat (Megalomys) from Barbados

Recently extinct insular populations of oryzomyine rice rats (Cricetidae: Sigmodontinae) are known from across the Lesser Antilles, but most remain undescribed. Historical records of a possible now-extinct endemic rodent from Barbados are supported by presence of oryzomyine remains in Late Quaternary sites on the island, including several pre-Columbian Holocene archaeological sites. The Barbados oryzomyine is described as Megalomys georginae sp. nov., and is most closely related to species from the nearby islands of Martinique and St. Lucia, M. desmarestii and M. luciae. These species all display a zygomatic plate with its posterior margin level with the alveolus of M1, a divided anterocone on M1, and distinct anterolophids and metaflexids on m2 and m3, and are recovered as a monophyletic clade in morphological-molecular and morphology-only analyses. The new species differs from other Megalomys species in having the labial accessory root of M1 absent, a reduced anteromedian flexus on M1, two roots on m2 and m3, a supratrochlear foramen on the humerus, and in its much smaller body size. Other extinct Caribbean oryzomyines are not recovered in a monophyletic clade with Megalomys.     

Biochemical and structural characterization of the Pak1-LC8 interaction

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser(88) inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding sequence compared with other characterized LC8 binding sequences. Together, these observations raise the question whether the Pak1/LC8 interaction is distinct (i.e. enabled by a unique interface independent of LC8 dimerization). Herein, we present results from biochemical, NMR, and crystallographic studies that show that Pak1 (residues 212-222) binds to LC8 along the same groove as canonical LC8 interaction partners (e.g. nNOS and BimL). Using LC8 point mutants K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding sequences and identify a key hydrogen bond network that compensates for the loss of the conserved glutamine in the consensus sequence. We also show that the target binding interface formed through LC8 dimerization is required to bind to Pak1 and precludes phosphorylation of LC8 at Ser(88). Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although these results define structural details of the Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they do not support the current model whereby Pak1 binds to and subsequently phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest that LC8 binding modulates Pak1 activity and/or nuclear localization.