Discrete charge calculations of potentiometric titrations for globular proteins: sperm whale myoglobin, hemoglobin alpha chain, cytochrome c.

The modified Tanford-Kirkwood theory of Shire et al. for intramolecular electrostatic interactions has been applied to hydrogen ion equilibria of sperm whale ferrimyoglobin, human hemoglobin α-chain and horse cytochrome c. The model employs two sets of parameters derived from the crystalline protein structures, first, the atomic coordinates of charged amino acid residues and, second, static accessibility factors to reflect their solvent exposure. In addition, a consistent set of intrinsic pK values (pK_int) for the individual groups is employed. The theoretical pK values at half-titration for individual groups in each protein correspond to the available observed pK values, and the theoretical titration curves compare closely with experimental potentiometric curves.     

Plasmid-determined beta-lactamase indistinguishable from the chromosomal beta-lactamase of Escherichia coli.

A plasmid, derived from a naturally occurring strain of Proteus mirabilis, conferred resistance to cephalosporins, apparently mediated by a beta-lactamase indistinguishable from that determined by the chromosomal gene of Escherichia coli K-12. There was evidence for a recombination event between the wild-type plasmid and a defective F factor (Fsp) in the Escherichia coli K-12 culture in which it was stored.     

The status of dwarf carnivores on Cozumel Island, Mexico

Cozumel Island in the Mexican Caribbean is inhabited by four carnivores, of which two, the Cozumel coati Nasua nelsoni and pygmy raccoon Procyon pygmaeus, are endemic species. The taxonomic status of a third carnivore, a dwarf gray fox Urocyon cinereoargenteus, is undetermined, but may deserve subspecific or species-level recognition. The fourth species, the kinkajou (Potos flavus), may be a recent introduction. We review the status of these carnivores, report our field observations and results of line transect and trapping efforts, discuss current threats to these taxa, and make recommendations for their conservation. A population density of 0.43 ± 0.27 coatis/km², and a total island population size of 150 ± 95 individuals, was estimated from 386 km of line transects in 1994–1995. Intensive trapping efforts (1479 trap-nights) in 2001 at multiple localities were unsuccessful. Pygmy raccoons were observed in the mangrove and coastal wetland areas of the island and in 2001 we captured 11 individuals with the same sampling efforts as for coatis (8.8 raccoons/1000 trap-nights). The gray fox is also apparently very rare on the island. While a few observations of the animals have been made (1984, 1994 and 2001), no animals were seen along transects and none were trapped. The primary threats to the persistence of these taxa include introduced congeners, introduced predators, parasite and disease spill-over from exotic animals, habitat fragmentation, hunting and collection as pets, and hurricanes. We suggest that the Cozumel coati, pygmy raccoon, and the Cozumel population of the gray fox be considered as Critically Endangered according to the IUCN classification system. Current conservation actions focusing on Cozumel carnivores are extremely limited. We recommend eradication of introduced species, maintenance of habitat connectivity, ex situ conservation programs, explicit public policies on land-use and sustainable development, public awareness campaigns, and continuous scientific research and monitoring.     

Diversity of anaerobic gut fungal populations analysed using ribosomal ITS1 sequences in faeces of wild and domesticated herbivores

Gut fungal-specific PCR primers have been used to selectively amplify the ITS1 region of gut fungal rDNA recovered from faeces of domestic and wild animals to investigate population diversity. Two different gel-based methods are described for separating populations of gut fungal rDNA amplicons, namely (1) denaturing gradient gel electrophoresis (DGGE) and (2) separation according to small size differences using Spreadex, a proprietary matrix for electrophoresis. Gut fungal populations were characterised by analysis of rDNA in faeces of seventeen domesticated and ten wild herbivores. Sequences derived from these gel-based characterisations were analysed and classified using a hidden Markov model-based fingerprint matching algorithm. Faecal samples contained a broad spectrum of fungi and sequences from five of the six recognised genera were identified, including Cyllamyces, the most recently described gut fungal genus, which was found to be widely distributed in the samples. Furthermore, four other novel groupings of gut fungal sequences were identified that did not cluster with sequences from any of the previously described genera. Both gel- and sequence- based profiles for gut fungal populations suggested a lack of geographical restriction on occurrence of any individual fungal type.     

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys₂His₂) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.     

Can genomics clarify the origins of Boreioglycaspis melaleucae in California,USA?

The Australian psyllid Boreioglycaspis melaleucae is a biological control agent of Melaleuca quinquenervia in Florida (USA) but was observed attacking M. quinquenervia trees in southern California (USA). Genotyping revealed the California population matched three of eight Australian haplotypes and all three Florida haplotypes. It remains unclear if the California psyllid population arrived directly from Australia or via Florida.     

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.