A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality

Methylmalonic aciduria is a human autosomal recessive disorder of organic acid metabolism resulting from a functional defect in the activity of the enzyme methylmalonyl-CoA mutase. Based upon the homology of the human mutase locus with the mouse locus, we have chosen to disrupt the mouse mutase locus within the critical CoA binding domain using gene-targeting techniques to create a mouse model of methylmalonic aciduria. The phenotype of homozygous knock-out mice (mut-/-) is one of early neonatal lethality. Mice appear phenotypically normal at birth and are indistinguishable from littermates. By 15 h of age, they develop reduced movement and suckle less. This is followed by the development of abnormal breathing, and all of the mice with a null phenotype die by 24 h of age. Urinary levels of methylmalonic and methylcitric acids are grossly increased. Measurement of acylcarnitines in blood shows elevation of propionylcarnitine with no change in the levels of acetylcarnitine and free carnitine. Incorporation of [14C]propionate in primary fibroblast cultures from mut-/- mice is reduced to approximately 6% of normal level, whereas there is no detectable synthesis of mut mRNA in the liver. This is the first mouse model that recapitulates the key phenotypic features of mut0 methylmalonic aciduria.     

Transcriptional regulation of the human TATA binding protein gene

The human TATA binding protein (TBP) locus consists of a functional domain of three closely linkedhousekeeping genes (TBP, PSMB1 (proteasomal C5 subunit), and PDCD2 (programmed cell death-2)) within a 50-kb interval at chromosome position 6q27. Here we demonstrate that a genomic clone spanning the 20-kb TBP gene, with 12 kb 5' and 3' flanking sequences, was fully functional in stable, transfected L-cells harboring a single copy of this transgene, including after long-term (60 day) culture in the absence of drug selective pressure. Furthermore, we were only able to detect DNaseI hypersensitive sites at the TBP and PSMB1 promoters present within this 44-kb fragment. Our data suggest that this 44-kb genomic region possesses genetic regulatory elements that not only drive ubiquitous expression of TBP but also negate chromatin and DNA methylation induced silencing, which is normally associated with transgenes stably integrated into tissue culture cells.     

Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette

There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human beta-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I-SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I-SceI site in the counterselection cassette is cut by I-SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants.     

Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function

Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADP-stimulated mitochondrial respiration in situ using saponin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADP-stimulated oxygen consumption and the dissociation constant (K(m)) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant role in mitochondrial positioning and respiratory function in cardiac and skeletal muscle.     

Invasive Italian wall lizards outcompete native congeneric species in finding food in a Y-maze

Though biological invasions constitute one of the biggest threats for global biodiversity, our understanding of the mechanisms that enable invasive species to outperform native species is still limited, especially, in terms of behavior. Most available studies have examined behavioral traits which favor invasive species on the later stages of invasion, however, our knowledge on earlier stages, namely, when alien species face novel environments and must exploit new resources, remains obscure. Here, we focus on one crucial behavioral trait, finding food. The Italian wall lizard (Podarcis siculus) has been widely introduced and established viable populations in S. Europe and N. America. We examined whether P. siculus has enhanced exploratory behavior and abilities to find food compared to two native congeneric species with which it may come in contact in the near future, an insular endemic (P. milensis) and a widely distributed lizard (P. erhardii). We performed a Y-maze experiment, in which we varied arm markings in a standard way to prevent learning. Podarcis siculus was more efficient than its congenerics in finding and consuming food. This exploitative superiority was persistent, more frequent and repetitive. Interesting behavioral differences were also detected within the native species. Some P. milensis individuals showed no interest in exploring the maze, while few P. erhardii individuals remained rather indifferent to food even after detecting it. Our results suggest that the invasive P. siculus displays behavioral traits that could provide better opportunities for survival in the new environment and thus facilitate establishment even in the presence of congenerics. This provides further support to the idea that behavior plays a crucial role in animal invasions.

     

Species diversity,endemism and conservation of the family Caryophyllaceae in Greece

The family Caryophyllaceae includes world-wide 86 genera and approximately 2100 species. Greece is one of its most important centres of diversity and endemism. A total of 428 Caryophyllaceae taxa are distributed in Greece, 161 of them being endemic to the Greek political territory. The endemic element represents approximately 5% of the global diversity of the family at the species level. The aim of this paper is to discuss the distribution patterns of the Greek endemic Caryophyllaceae, as well as those with a limited distribution range to the neighbouring areas of the Balkans and Anatolia, on the basis of the phytogeographical regions of Greece in order to identify the important regions for their conservation. The majority of the Greek endemic Caryophyllaceae (64.6%) are distributed in only one single phytogeographical region, or even a smaller area indicating the extremely restricted distribution ranges of the endemic plants in Greece. Actually 83 Greek endemic Caryophyllaceae can be grouped on cytotaxonomic criteria. Most of them belong to the category of schizoendemics (91.6%), indicating that the endemism of Caryophyllaceae in Greece has mainly originated in an active way. Cluster analysis has been used to classify the phytogeographical regions according to their floristic similarities. Two iterative complementarity methods were used to evaluate the importance of each phytogeographical region in the conservation of the endemic Caryophyllaceae in Greece. Peloponnisos, Kriti-Karpathos and Sterea Ellas are the most important phytogeographical regions in this respect, followed by North Central and North East. When adding the Balkan-Aegean-Anatolian endemics to the analysis, Peloponnisos, North Central, Kriti-Karpathos, North East and Eastern Aegean result as the most important areas. In every case, an elevated number of sites are required for the conservation of Caryophyllaceae in Greece, reflecting the great dissimilarities in the floristic composition of the various phytogeographical regions. The results provided by the different methods are compared. A catalogue of the Greek endemic Caryophyllaceae is appended.     

Questioning the effectiveness of the Natura 2000 Special Areas of Conservation strategy: the case of Crete

Aim This study examines the effectiveness of the selected ‘network’ of Natura 2000 Special Areas of Conservation (SACs) at a regional scale in Greece, in terms of its representativeness of plant biodiversity. Location The island of Crete is used as a case study because it is considered to be one of the 10 hotspots for biodiversity in the Mediterranean Basin. Methods Hotspot analysis and complementarity algorithms are used to define priority areas for conservation and calculate their spatial overlap with the Natura 2000 SACs in Crete. Results The various categories of hotspots contain subsamples of plant categories, used for their definition. Spatial overlap among different categories of hotspots, areas of complementary diversity and Natura 2000 SCAs is low. Main conclusions The results show that the Natura 2000 SACs ‘network’ in Crete seems insufficient to ensure satisfactory representation of the regional plant biodiversity elements.