Isolation of a 25-kDa protein binding to a curved DNA upstream the origin of the L strand replication in the rat mitochondrial genome

The presence of a curved DNA sequence in the gene for the NADH-dehydrogenase subunit 2 of rat mitochondrial genome, upstream from the origin of the light strand replication have been demonstrated through theoretical analysis and experimental approaches. Gel retardation assays showed that this structure makes a complex with a protein component extracted from the mitochondrial matrix. The isolation and purification of this protein is reported. With a Sepharose CL-6B and magnetic DNA affinity chromatography a polypeptide was purified to homogeneity having 25-kDa mass as shown by gel electrophoresis. To functionally characterize this protein, its capability to bind to other sequences of the homologous or heterologous DNA and to specific riboprobes was also investigated. A role for this protein as a trans-acting agent required for the expression of the mammalian mitochondrial genome is suggested

The observed oogenesis impairment in greater amberjack Seriola dumerili (Risso, 1810) reared in captivity is not related to an insufficient liver transcription or oocyte uptake of vitellogenin

The greater amberjack Seriola dumerili is an excellent candidate for the Mediterranean aquaculture, due to its large body size and high growth rate, as well as its high flesh quality and commercial value worldwide. For its successful incorporation in the aquaculture industry, an in-depth understanding of the reproductive function of the species under rearing conditions is necessary, since completion of oogenesis in captivity is currently a bottleneck for the commercial production of the species. Liver and ovary samples from wild and captive-reared greater amberjack females were collected at three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). The cDNAs of three vitellogenins (VtgA, VtgB and VtgC) were partially sequenced and a qRT-PCR for their expression was used to compare ovarian maturity stage and liver vitellogenin transcript levels between wild and captive-reared individuals. An extensive atresia of late vitellogenic follicles, which prevented any further oocyte development and spawning was observed in captive-reared individuals during the ADVANCED phase. The expression levels of the three vitellogenins, as well as the amount of yolk globules in vitellogenic oocytes, did not differ significantly between captive-reared and wild females, indicating that the observed oogenesis impairment in greater amberjack reared in captivity was not related to an insufficient liver synthesis or a reduced oocyte uptake of vitellogenin

Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity.

Eukaryotic cells contain a population of mitochondria, variable in number and shape, which in turn contain multiple copies of a tiny compact genome (mtDNA) whose expression and function is strictly coordinated with the nuclear one. mtDNA copy number varies between different cell or tissues types, both in response to overall metabolic and bioenergetics demands and as a consequence or cause of specific pathological conditions. Here we present a novel and reliable methodology to assess the effective mtDNA copy number per diploid genome by investigating off-target reads obtained by whole-exome sequencing (WES) experiments. We also investigate whether and how mtDNA copy number correlates with mitochondrial mass, respiratory activity and expression levels. Analyzing six different tissues from three age- and sex-matched human individuals, we found a highly significant linear correlation between mtDNA copy number estimated by qPCR and the frequency of mtDNA off target WES reads. Furthermore, mtDNA copy number showed highly significant correlation with mitochondrial gene expression levels as measured by RNA-Seq as well as with mitochondrial mass and respiratory activity. Our methodology makes thus feasible, at a large scale, the investigation of mtDNA copy number in diverse cell-types, tissues and pathological conditions or in response to specific treatments.This work was supported by Ministero dell'Istruzione, Università e Ricerca (projects PRIN-2009, Micromap [PON01_02589], Virtualab [PON01_01297]) and by Consiglio Nazionale delle Ricerche (progetto strategico “Medicina personalizzata”, progetto strategico “Invecchiamento”, progetto bandiera “Epigen”)

Pseudogenes in metazoa: Origin and features

The complete genome sequences with their annotations are a considerable resource in biology, particularly in understanding the global structure of the genetic material at the molecular level. The reason why some eukaryotic genomes contain large quantities of apparently unnecessary DNA, namely pseudogenes, while others seem to invest in more efficient thinning processes or are equipped with protection systems against parasitic elements still remains a mystery. Several genome-wide surveys have been undertaken to identify pseudogenes in the completely sequenced genome, bringing to light some differences both in their amount and distribution. Since pseudogenes are important resources in evolutionary and comparative genomics — as ‘molecular fossils’ — in this paper, a survey on the origins, features, abundance and localisation of the different pseudogenes is reported. As an example of genes producing processed pseudogenes, some experimental data obtained in the authors’ laboratories from the study of a nuclear gene coding for the mitochondrial transcription factor A (mtTFA), a key regulator of mitochondrial biogenesis, are also reported

Transient overexpression of an alternative spliced isoform of mitochondrial transcription factor A (Δ5TFAM) affects mitochondrial transcription in the H1299 cell line

Mitochondrial transcription factor A (Tfam) is necessary for both transcription and maintenance of mitochondrial DNA, it is abundant enough to wrap the entire mtDNA and thus organizes a protein-DNA complex. The gene is estimated to span about 10 kb in mouse, in human and in rat. The sequence of the human gene contains an open reading frame of 741 bp over seven exons and encodes a protein with a molecular weight of 25 kDa. A short mRNA of Tfam, lacking exon 5 (05Tfam), was found to be widely distributed in human tissues, representing 30% of the total Tfam transcript pool. Changes in the primary structure of the protein can alter the binding propertíes of proteins, influence their intracellular localization and modify their enzymatic activity and/or protein stability by diverse mechanisms as well as other characteristics (Stamm et al., 2005). 45Tfam isoform localization and its effect on mitochondrial transcription were studied. To analyze the localization of 05Tfam isoform a chimeric protein consisting of A5Tfam and of the green fluorescent protein GFP as visualized tag has been expressed. In vitro expression was performed by using the TNT Quick Coupled Transcription/Translation System (Promega). Proteins obtained were observed by immunoblotting. To study the effects of the alternative spliced 05Tfam on the transcriptional activity, transient overexpression of either full length Tfam or 05Tfam in H1299 celi was carried out, and reactions of Sybr Green Reaf time-PCR were then performed to observe the behaviour of Tfam, 05Tfam and of the mitochondrial transcript COI. The results reported show that mitochondrial transcription increases eìther after full length Tfam either after 05Tfam transfection although less efficiently for the second one. These results agree with the smaller capability of 05Tfam to bind mtDNA in comparison witthe smaller capability of 05Tfam to bind mtDNA in comparison with the full length isoform