Proteinase K Processing of Rabbit Muscle Creatine Kinase

Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A₃₂₈ and A₃₂₄, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A₃₂₈–K₃₈₀). However, the C-terminal end of the K1 fragment is not A₃₂₇ as expected, but D₃₂₅. Thus, the amino acids residues T₃₂₆ and A₃₂₇ have been eliminated by the protease.     

Diagnosis of defects in oxidative muscle metabolism by non-invasive tissue oximetry

The dynamics of oxygen delivery and utilization are examined in a variety of mitochondrial disorders during rest, exercise and post exercise. We used a non-invasive optical technique to measure the oxygen consumption in the exercising limb in normal subjects and 5 patients with cytochrome c oxidase deficiency. We also examined 6 patients with MELAS and MERRF syndrome. We measured near-infrared spectra of hemoglobin in the gastrocnemius muscle during treadmill exercise. Normal subjects demonstrated a sustained deoxygenation during exercise, indicating an efficient utilization of delivered oxygen. Patients with cytochrome c oxidase deficiency demonstrated consistent oxygenation during exercise indicating an under utilization of delivered oxygen. Patients with MELAS and MERRF syndrome showed similar under utilization of oxygen during exercise. Non-invasive tissue oximetry during exercise demonstrates specific abnormalities in a variety of mitochondrial disorders, indicating abnormal oxygen utilization, and will be a useful addition to the clinical investigation of such disorders. (Mol Cell Biochem 174: 7–10, 1997)     

Histidine and histamine metabolism in rat enterocytes

We have shown that the Metabolic Control Analysis (MCA) can explain the threshold effect observed in the expression of mitochondrial diseases [8]. As a matter of fact, the effect of a specific inhibitor on the flux of O2 consumption mimics a defect in a step of oxidative phosphorylation. The observed threshold is correlated to the value of the control coefficient of the inhibited step.For this reason, we have studied the repartition of the control coefficients of different steps in oxidative phosphorylation on various tissues (liver, kidney, brain, skeletal muscle and heart). We discuss the results in terms of metabolic control theory and provide a possible explanation for the heterogeneous phenotype of those pathologies. We present the double threshold hypothesis of both a threshold in the energy demand of a tissue and in the energy supply by oxidative phosphorylation. (Mol Cell Biochem 174: 143–148, 1997)     

SPECIATIONAL HISTORY IN A DIVERSE CLADE OF HABITAT-SPECIALIZED SPIDERS (ARANEAE: NESTICIDAE: NESTICUS): INFERENCES FROM GEOGRAPHIC-BASED SAMPLING

This paper summarizes the results of an initial effort to reconstruct the speciational history of cave spiders (Nesticus) from the southern Appalachian Mountains of eastern North America. The Appalachian Nesticus fauna includes a large series of about 30 species distributed across islandlike cave and montane habitats. Many of the species are geographically restricted; all of the species are found in allopatry. Observed patterns of morphological variation and biogeographic evidence suggest that species diversification in this lineage may have occurred recently, perhaps in response to Pleistocene climatic fluctuations. To address questions about the spatial and temporal dynamics of Nesticus speciation, while accounting for potential phylogenetic difficulties, I have gathered nuclear and mitochondrial DNA sequences for a sample of individuals from 81 populations representing 28 Nesticus species. Analyses of these data indicate that considerable genetic divergence exists within and among currently recognized morphological species. Consistent with relatively deep species divergences, most of which likely predate the Pleistocene, is a prevailing pattern of phylogenetic concordance between taxonomic species and monophyletic gene tree lineages. The few deviations from monophyly detected can be tentatively attributed to a peripatric mode of speciation. Although species limits as inferred by the molecular data are generally concordant with patterns of morphological continuity and discontinuity in genitalia, there is evidence to suggest that cryptic phylogenetic lineages exist within some morphologically continuous units. This observation, in combination with the general depth of species lineages, makes any argument about rapid evolution in Nesticus genitalic characteristics unnecessary.     

A CONTACT ZONE WITH NONCOINCIDENT CLINES FOR SEX-SPECIFIC MARKERS IN THE FIELD VOLE (MICROTUS AGRESTIS)

A field vole (Microtus agrestis) population characterized by unique mitochondrial DNA (mtDNA) and Y chromosome markers occurs in southwest Sweden. A contact zone between this Lund (Lu) population and a standard (St) population was examined with two sex-specific genetic markers. The field vole mtDNA and Y chromosome clines display a remarkable lack of coincidence, rarely observed in contact zones. The cline width for both markers is about 50 km, but the two clines are displaced from each other: the mtDNA cline is found in the central part of the study area, whereas the cline for the Y chromosome is located in the eastern part. Thus, the absolute width of the Lu-St zone spans about 95–110 km. As a result of the cline shift, all male hybrids carry the Lu-Y chromosome and St-type mtDNA. The other possible male hybrid class is lacking. The distinct noncoincidence of the mtDNA and Y chromosome clines is most likely explained by selection against male hybrids with the St-Y chromosome and Lu-mtDNA. It is possible that incompatibilities between the maternal genome of Lu-type animals and the paternal genome of St-type individuals exist. However, alternative explanations based on neutrality or selective advantage cannot be totally dismissed.     

The retrotransposon RTip1 is integrated into a novel type of minisatellite, MiniSip1, in the genome of the common morning glory and carries another new type of minisatellite, MiniSip2

 Transposable elements have often been discovered as new insertion sequences in known genes, and minisatellites are often employed as molecular markers in diagnostic and mapping studies. We compared the genes for flower pigmentation in a line of the common morning glory bearing fully colored flowers with those in two anthocyanin ^flaked mutable lines producing variegated flowers and found RFLPs at the region of the ANS gene for anthocyanin biosynthesis. The DNA rearrangements detected by the RFLPs are due to integration of a novel type of minisatellite, MiniSip1, having a long LTR retrotransposon, RTip1, inserted in the mutable lines. The structural analysis of the rearranged region revealed that the 12.4-kb RTip1 element is flanked by 5-bp target duplications within the MiniSip1 sequence and contains two LTR sequences of about 590 bp, a primer binding site for tRNA^Lys, a typical polypurine tract and another new type of minisatellite, MiniSip2. Since no long open reading frame corresponding to the gag and pol genes was found, RTip1 appears to be a defective Ty3/gypsy-like element. Interestingly, the 269-bp-long MiniSip1 element comprises two alternating motifs of 41 bp and 19 bp, whereas the 962 bp long MiniSip2 element consists of two partially alternating motifs of 86 bp and 90 bp which are partially homologous to each other. Possible evolutionary processes that may have generated the rearranged structure at the ANS gene region are also discussed. Received: 25 April 1997 / Accepted: 16 May 1997     

Mitochondrial DNA variation and the evolutionary history of chromosome races of collared lemmings (Dicrostonyx) in the Eurasian Arctic

Collared lemmings (Dicrostonyx) demonstrate extensive chromosome variation along their circumpolar distribution in the high Arctic. To reveal the history of this genus and the origin of chromosome races in the Palearctic, we studied the geographical pattern of mtDNA variation in lemmings from 13 localities by using eight tetranucleotide restriction enzymes. The main split in mtDNA phylogeny is at the Bering Strait and corresponds to the main chromosome division between the Beringian and the Eurasian groups of karyotypes. Nucleotide divergence estimate of 6.8% suggests that, despite the Bering Land Bridge, Palearctic and Nearctic forms have been separated since the mid-Pleistocene. Five distinct phylogenetic groups of mtDNA haplotypes, with average divergence of 1.5%, corresponding to geographical regions, were found along the Palearctic coast. Low nucleotide and haplotype diversity and a star-like phylogeny within phylogeographical groups of haplotypes suggest regional bottleneck events in the recent past, most probably due to warming events during the Holocene. There is congruence between phylogeographical pattern of mtDNA variation and geographical distribution of chromosome races; 69% of the total mtDNA variation is allocated among chromosome races. This congruence implies that historical events such as fragmentation and allopatric bottleneck events have been important for the origin of chromosome races. However, historical factors do not explain the fixed autosome fusions found to distinguish certain populations.     

Mitochondrial DNA variation in Spanish populations of Ceratitis capitata (Wiedemann) (Tephritidae) and the colonization process

Abstract: Mitochondrial DNA variation in the Mediterranean fruit fly Ceratitis capitata (Wiedemann, Diptera: Tephritidae) was studied in three natural populations from southern, central and eastern Spain by means of restriction fragment length polymorphisms using 22 restriction endonucleases. Nine different haplotypes were found based upon the restriction patterns of the seven polymorphic endonucleases, providing a measure of discrimination between populations (N_ST = 0.2462, F_ST = 0.154). The observed distribution of haplotypes, corroborated by a parsimonious unrooted tree, suggests an ancient origin for haplotype VII, and a first step in the colonization of the Iberian Peninsula and subsequently the northern and eastern Mediterranean basin, through the Straits of Gibraltar. No relationship can be established between the colonization process for Europe and America.     

Historical interdrainage dispersal of eastern rainbowfish from the Atherton Tableand, north-eastern Australia

The widespread distribution of the eastern rainbow fish Melanotaenia splendida splendida throughout the isolated headwaters of the rivers on the Atherton Tableland, north-eastern Australia, suggests multiple colonization events from the eastern lowlands via each respective river channel, or a single colonization event on to the tableland with subsequent dispersal between the headwaters. To explore the likely processes that resulted in the current distribution on the tableland, two models of gene flow were tested: (a) the hierarchical gene flow model that tests the hypothesis for contemporary gene flow via stream channels and (b) the stepping stone model that tests for dispersal between streams. Neither of these models explained the observed genetic structure, adequately. However, there is support for extensive historical dispersal across the headwaters of the isolated drainages. If this dispersal followed a single colonization event, the subsequent range expansion could have facilitated a rapid rise in population size due to an increase in suitable habitat. The genetic data indicates an eight-fold increase in population size c. 100 thousand years ago.