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Kae1 is a subunit of the highly evolutionarily conserved KEOPS/EKC complex, which is involved in universal (t6A

The northern pike (Esox lucius L.) is a commercially important fish that forms freshwater and brackish populations. While the first ecotype is still quite abundant, the second has suffered rapid declines in recent years. Populations inhabiting the Baltic coastal waters of Poland have been supported by stocking programs since the late 1990s. This study describes a multiplex microsatellite (msDNA) assay consisting of 12 loci divided conveniently into two amplification sets (Elu78, Elu87, B451, Elu37, B457, B16, B25, and Elu19, Elu76, B422, Elu2, Elu7). The assay was optimized for genetic analyses of freshwater and brackish populations. These multiplexes were successfully executed to obtain the genetic profiles of 668 individuals from 16 populations (Poland and Germany). The average number of alleles was equal to 10.4, whereas the observed heterozygosity per locus ranged from 0.18 to 0.89, with the average value of 0.64. The probability of the identity of the marker sets indicated the high power of identification of unique genotypes. Therefore, this molecular tool can be used to describe the genetic variability of populations, select the proper source of breeding material, and monitor the progress of stocking efforts in genetic conservation projects of this species.

Development of the gene engineering techniques has raised worries that they will be used for construction of organism endangering humans and environment. In 1975 at the Asilomar conference, geneticists from many countries decided that genetic engineering brings more benefits than threats. In last years a new CRISPR-Cas technique emerged . It allows to make the precise changes in genomes, e.g. to inactivate particular genes or to replace mutated genes by their wild-type alleles. Inactivation in mice of genes corresponding to those whose mutations cause the genetic diseases in man allows to get model organisms for studying the etiology of given disease and for working out the methods of its curing. This technique can be applied for repairing genes whose mutations result in metabolic diseases and cancer. Some voices were raised that the technique can be potentially used for the "improvement" of man, what would create many ethical and social problems. Geneticists, ethicists and lawyers gathered in 2015 at the Washington conference, discussed these problems and proposed rules for their solving.Opracowanie technik inżynierii genetycznej wywołało obawy iż będą one wykorzystane do konstruowania organizmów zagrażających człowiekowi i środowisku. W roku 1975, na konferencji w Asilomar w Kalifornii, genetycy z wielu krajów uznali, że ze stosowaniem inżynierii genetycznej wiąże się więcej potencjalnych korzyści niż zagrożeń. W ostatnich latach pojawiła się nowa technika CRISPR-Cas, pozwalająca na dokonywanie precyzyjnych zmian w genomach organizmów, np. dezaktywowanie określonych genów lub wstawianie prawidłowych. Dezaktywując u myszy odpowiedniki genów, których mutacje powodują choroby genetyczne człowieka otrzymujemy modele pozwalające na poznanie etiologii danej choroby i opracowanie metod jej leczenia. Technikę tę możemy też wykorzystać do naprawy zmutowanych genów, odpowiedzialnych za choroby metaboliczne i nowotworowe człowieka. Pojawiły się obawy, że może być ona wykorzystana do „udoskonalania” człowieka, co rodziłoby szereg problemów etycznych i społecznych. Genetycy, etycy i prawnicy zebrani na konferencji w Waszyngtonie w 2015 roku przedyskutowali te problemy i zaproponowali zasady stosowania techniki CRISP-Cas.

Archeological and genetic evidence suggest that all domestic cats derived from the Near Eastern wildcat (Felis silvestris lybica) and were first domesticated in the Near East around 10,000 years ago. The spread of the domesticated form in Europe occurred much later, primarily mediated by Greek and Phoenician traders and afterward by Romans who introduced cats to Western and Central Europe around 2000 years ago. We investigated mtDNA of Holocene Felis remains and provide evidence of an unexpectedly early presence of cats bearing the Near Eastern wildcat mtDNA haplotypes in Central Europe, being ahead of Roman period by over 2000 years. The appearance of the Near Eastern wildcats in Central Europe coincides with the peak of Neolithic settlement density, moreover most of those cats belonged to the same mtDNA lineages as those domesticated in the Near East. Thus, although we cannot fully exclude that the Near Eastern wildcats appeared in Central Europe as a result of introgression with European wildcat, our findings support the hypothesis that the Near Eastern wildcats spread across Europe together with the first farmers, perhaps as commensal animals. We also found that cats dated to the Neolithic period belonged to different mtDNA lineages than those brought to Central Europe in Roman times, this supports the hypothesis that the gene pool of contemporary European domestic cats might have been established from two different source populations that contributed in different periods.

The present distribution of many species is a result of climatic changes during the Pleistocene and human activity. The impact of climate has been accompanied by restrictions of populations into re ...

In Aspergillus nidulans, nitrogen and carbon metabolism are under the control of wide-domain regulatory systems, including nitrogen metabolite repression, carbon catabolite repression and the nutrient starvation response. Transcriptomic analysis of the wild type strain grown under different combinations of carbon and nitrogen regimes was performed, to identify differentially regulated genes. Carbon metabolism predominates as the most important regulatory signal but for many genes, both carbon and nitrogen metabolisms coordinate regulation. To identify mechanisms coordinating nitrogen and carbon metabolism, we tested the role of AreB, previously identified as a regulator of genes involved in nitrogen metabolism. Deletion of areB has significant phenotypic effects on the utilization of specific carbon sources, confirming its role in the regulation of carbon metabolism. AreB was shown to regulate the expression of areA, tamA, creA, xprG and cpcA regulatory genes suggesting areB has a range of indirect, regulatory effects. Different isoforms of AreB are produced as a result of differential splicing and use of two promoters which are differentially regulated by carbon and nitrogen conditions. These isoforms are likely to be functionally distinct and thus contributing to the modulation of AreB activity., AreB is a wide-domain regulator mediating a transcriptional response to changes in both nitrogen and carbon conditions.

Examination of faunal remains from archaeological sites facilitates investigation of the history of human–animal interactions, domestication-related pathways and subsistence strategies. In South America, archaeological remains of dogs are rare and confined mostly to the Andean region. Such remains are often fragmentary and can be difficult to identify, particularly in the presence of closely related canine species. In this study, we performed ancient DNA analysis and radiocarbon dating to verify the species and age of five putative pre-Columbian dog remains excavated from the Formative to Tiwanaku contexts at four archaeological sites in Bolivia. Phylogenetic analysis and radiocarbon dating revealed that only one sample was of a pre-Columbian dog, whereas two samples were identified to be of contemporary dogs and two of South American foxes. Our results underline the need for verifying species assignment and age, particularly of fragmentary remains, even if they are found at seemingly undisturbed archaeological contexts.

Archaeological and genetic evidence suggest that all domestic cats derive from the Near Eastern wildcat (Felis silvestris lybica) and were domesticated twice, first in the Near East around 10 000 years ago and for the second time in Ancient Egypt ca. 3 500 years ago. The spread of the domesticated form in Europe occurred much later, primarily mediated by Greek and Phoenician traders and afterwards by Romans who introduced cats to Western and Central Europe around 2 000 years ago. We investigated mtDNA of Holocene Felis remains and provide evidence of an unexpectedly early presence of cats bearing the Near Eastern wildcat mtDNA haplotypes in Central Europe, being ahead of Roman Period by over 2 000 years. The appearance of the Near Eastern wildcats in Central Europe coincide with the peak of Neolithic settlement density, moreover most of those cats belonged to the same mtDNA lineages as those domesticated in the Near East. Thus, although we cannot fully exclude that the Near Eastern wildcats appeared in Central Europe as a result of introgression with European wildcat, our findings strongly support the hypothesis that the Near Eastern wildcats spread across Europe together with the first farmers, perhaps as commensal animals. We also found that cats dated to the Neolithic period belonged to different mtDNA lineages than those brought to Central Europe in Roman times, this supports the hypothesis that the gene pool of contemporary European domestic cats might have been established from two different source populations that contributed in different periods.

Human-induced hybridization between previously allopatric species deserves special attention from conservationists. The European whitefish (Coregonus lavaretus), native fish species in Poland, hybridizes with another coregonid fish—peled (Coregonus peled) due to artificial fertilization in the hatcheries. The latter species is native to Siberian waters and was introduced into Poland in 1966. The offspring of these two species are fertile, and interspecific hybrids have been shown to be present in certain Polish lakes. In this study, we estimate the level of peled introgression into the whitefish gene pool and verify the presence of pure whitefish populations in the four Polish lakes. Genetic analysis of 12 microsatellite loci and ITS 1 (internal transcribed spacer 1) fragment sequences revealed limited introgression. Bayesian admixture analysis showed that only one lake harbored an admixed population. All six admixed individuals possessed European whitefish mtDNA haplotypes. This study is the first comprehensive investigation of whitefish genetic diversity in Poland and revealed very limited introgression of peled. Obtained results showed that pure whitefish populations are still present in Poland and represent appropriate source for supportive breeding programs.

Understanding mutation rates can greatly extend the utility of population and conservation genetic analyses. Herein, we present an estimate of genome-wide microsatellite mutation rate in Atlantic sturgeon (Acipenser oxyrinchus) based on parent-offspring transmission patterns. We screened 307 individuals for parentage and mutation-rate analysis applying 43 variable markers. Out of 13228 allele transfers, 11 mutations were detected, producing a mutation rate of 8.3 × 10-4 per locus per generation (95% confidence interval: 1.48 × 10-3, 4.15 × 10-4). Single-step mutations predominated and there were trends toward mutations in loci with greater polymorphism and allele length. Two of the detected mutations were most probably cluster mutations, being identified in 12 and 28 sibs, respectively. Finally, we observed evidences of polyploidy based on the sporadic presence of 3 or 4 alleles per locus in the genotyped individuals, supporting previous reports of incomplete diploidization in Atlantic sturgeon.

The migration of Ursus ingressus from Southern to Western Europe, where it replaced native forms of Ursus spelaeus, is well documented. However, its occurrence in Central and Eastern European countries is unexplored. We have characterized the cave bear population inhabiting the Sudetes Mountains during the Late Pleistocene. DNA analyses were performed on 39 cave bear specimens from the Niedźwiedzia Cave. Ten successfully yielded the 254-bp sequences of an mtDNA control region. Phylogenetic analyses allowed assignment of nine samples to the U. ingressus haplogroup and one to brown bear (Ursus arctos). The mtDNA haplotypes of U. ingressus formed a single cluster and differed from haplotypes obtained for cave bears from other excavation sites in Europe. Uranium–thorium and radiocarbon dating of selected samples has shown that U. ingressus appeared in the Sudetes region at least 80,000 years ago. This finding may suggest that the appearance of U. ingressus in this area resulted from migration that occurred earlier than the colonization of the Alps and Swabian Jura. It is possible that migration of U. ingressus proceeded along the Carpathian and Sudetes arc.

We performed genetic analyses of three Baltic eelgrass (Zostera marina) populations in Puck Bay (PB), Cudema Bay (CB) and Greifswalder Bodden (GB). The aim of this study was to identify the eelgrass population genetically closest to that from the PB, which could potentially serve as a reservoir for the restoration of the underwater meadows in this bay, seriously degraded in the past. We applied a 12-microsatellite assay to test the genetic distance between the target eelgrass populations. We found that the allelic richness values of the GB, PB and CB populations were 2.25, 3.77 and 3.50 respectively. The genetic diversity found in GB was low and could be explained by the population’s history, whereas the diversity of CB was higher than expected in a population located at the edge of the species’ range. Analyses of genetic differentiation and structure showed that of the three populations studied, PB and CB were closer to each other than to the GB population. The reasons for this differentiation in eelgrass populations and the implications of the results of their genetic analysis on the planned restoration of the PB populations are discussed.

The Antiquity of Southeastern Europe Research Center, University of Warsaw, Krakowskie Przedmiescie 32,00-927 Warsaw, PolandKEY WORDS Acchaymarca; ancient DNA; microsatellites; Puca; Tompullo 2ABSTRACT The common practice of resettlementand the development of administrative and ceremonialsystems shaped the population landscape of the Andeanregion under the Inca rule. The area surrounding Coro-puna and Solimana volcanoes, in the Arequipa region(Peru), carried a high-density, multiethnic population.We studied the genetic variation among three pre-Columbian populations from three functionally diversearchaeological sites excavated in this region. By analyz-ing the genetic composition of a large ceremonial center(Acchaymarca), an isolated pastoral settlement (Tom-pullo 2), and an agricultural settlement characterizedby architectural features rare in the region (Puca), weinvestigated the patterns of population movements andthe distribution of genetic diversity. We obtained mito-chondrial DNA sequences for 25 individuals and auto-somal microsatellite profiles for 20 individuals fromAcchaymarca and Puca sites. These were comparedwith previously published genetic data for Tompullo 2and other pre-Columbian populations. We found differ-ences among the genetic portraits of the three popula-tions, congruent with the archaeologically describedfunctions and characteristics of the sites. The Acchay-marca population had the highest genetic diversity andpossessed the lowest number of unique mtDNA haplo-types. The Tompullo 2 population exhibited the lowestlevel of genetic diversity. The Puca population was dis-tinct from the other two populations owing to a highfrequency of haplogroup A haplotypes, what potentiallyexplains the non-local character of the burial architec-ture. Our analyses of microsatellite data suggest thatgene flow between sites was mostly mediated byfemales, which is consistent with ethnohistorical knowl-edge of the social organization of the pre-Columbiancommunities. Am J Phys Anthropol 000:000–000,2014.

We have developed a multiplex assay covering 16 microsatellite loci, amplified in four polymerase chain reaction (PCR) assays, and loaded on the ABI DNA Analyzer in two separate panels. The assay was tested on 603 individuals originating from wild populations and hatchery stocks of Atlantic sturgeon. The assay was also tested on 12 individuals of European sturgeon and appeared to be almost equally useful. The multiplex assay designed in this study can be successfully applied in studies requiring high genetic resolution, such as relatedness analysis, selective breeding programs, and stock identification of Atlantic sturgeon. Electronic supplementary material The online version of this article (doi:10.1007/s13353-014-0216-y) contains supplementary material, which is available to authorized users.

Aim Migrants of the Atlantic sturgeon, Acipenser oxyrinchus, from North America are thought to have founded the Baltic sturgeon population during the Little Ice Age around 1200 years ago, replacing the European sturgeon, Acipenser sturio. To test this hypothesis and to further elucidate the colonization of the Baltic Sea by A. oxyrinchus, we carried out DNA analyses of ancient and contemporary populations of both species. Location We analysed DNA from 188 specimens of sturgeons collected from archaeological sites and museums in Poland and of 225 contemporary specimens from North American and European populations. Methods Several mitochondrial DNA fragments were sequenced and eight microsatellite loci were genotyped for species identification, polymorphism and population structure analyses. Approximate Bayesian computation was used to estimate when the Baltic Sea was colonized. Results Of 125 ancient sturgeon specimens from the Baltic Sea, only four were classified as A. sturio, the remainder being A. oxyrinchus oxyrinchus. The ancient A. o. oxyrinchus population over two different time periods was highly polymorphic and genetically distant from contemporary populations of this taxon. The time of entry into the Baltic Sea was estimated to be 4000–5000 years ago. We also detected introgression of A. sturio into the A. o. oxyrinchus gene pool, caused by a prior hybridization event. Main conclusions For the past 2000 years at least, A. o. oxyrinchus has been the dominant sturgeon in the Baltic Sea, indicating a much earlier origin than previously suggested. The most similar extant sturgeon populations to the extinct Baltic stock are those from the St John and St Lawrence rivers in Canada. These populations should be considered the best source of breeding material for the ongoing sturgeon restitution programmes in Poland and Germany.

Summary The two sister species of Atlantic and European sturgeon are difficult to distinguish solely by morphological traits, especially in their early life stages. A simple PCR-RFLP based test was developed, enabling easy identification of specimens belonging to both species. The application of two restriction enzymes (BtsI and BsrDI) allowed the clear and unambiguous discrimination of 132 specimens of Atlantic and European sturgeon. The test should prove particularly useful with regard to the sturgeon restitution programs currently underway on both sides of the Atlantic Ocean.

Differential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well-characterized example is glutamine signalled degradation of specific transcripts in Aspergillus nidulans. In the case of areA, which encodes a wide-domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3′ UTR. Utilizing this RNA sequence we isolated RrmA, an RNA recognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization of niaD mRNA. However, nitrogen starvation was shown to act independently of RrmA in stabilizing certain transcripts. RrmA was also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmA mutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization of eifE and dhsA mRNA. eifE encodes eIF5A, a translation factor within which a conserved lysine is post-translationally modified to hypusine, a process requiring DhsA. Intriguingly, for specific transcripts RrmA mediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts.

Recent palaeogenetic studies indicate a highly dynamic history in collared lemmings (Dicrostonyx spp.), with several demographical changes linked to climatic fluctuations that took place during the last glaciation. At the western range margin of D. torquatus, these changes were characterized by a series of local extinctions and recolonizations. However, it is unclear whether this pattern represents a local phenomenon, possibly driven by ecological edge effects, or a global phenomenon that took place across large geographical scales. To address this, we explored the palaeogenetic history of the collared lemming using a next‐generation sequencing approach for pooled mitochondrial DNA amplicons. Sequences were obtained from over 300 fossil remains sampled across Eurasia and two sites in North America. We identified five mitochondrial lineages of D. torquatus that succeeded each other through time across Europe and western Russia, indicating a history of repeated population extinctions and recolonizations, most likely from eastern Russia, during the last 50 000 years. The observation of repeated extinctions across such a vast geographical range indicates large‐scale changes in the steppe‐tundra environment in western Eurasia during the last glaciation. All Holocene samples, from across the species' entire range, belonged to only one of the five mitochondrial lineages. Thus, extant D. torquatus populations only harbour a small fraction of the total genetic diversity that existed across different stages of the Late Pleistocene. In North American samples, haplotypes belonging to both D. groenlandicus and D. richardsoni were recovered from a Late Pleistocene site in south‐western Canada. This suggests that D. groenlandicus had a more southern and D. richardsoni a more northern glacial distribution than previously thought. This study provides significant insights into the population dynamics of a small mammal at a large geographical scale and reveals a rather complex demographical history, which could have had bottom‐up effects in the Late Pleistocene steppe‐tundra ecosystem.

The Emine-Bair-Khosar Cave (EBK), situated on the northern edge of the Lower Plateau of the Chatyrdag Massif (Crimean Mountains) is rich in palaeontological material accumulated over most of the Pleistocene. mtDNA was isolated from bones of three specimens of red deer (Cervus elaphus) dated to the late Pleistocene (MIS 3). These are the first ancient DNA sequences obtained for this species. The position of the three red deer individuals on the phylogeographic tree is based on mtDNA sequences of contemporary representatives of the Cervinae inhabiting the Northern Hemisphere. The results confirm the notion that the Crimean Peninsula was the north-easternmost refugium in Europe, and that during and after the Late Pleistocene it played a major role in recolonisation and dispersal of temperate species in the whole Eurasian continent.

The total number of bacteria and culturable bacteria in Adélie penguin (Pygoscelis adeliae) guano was determined during 42 days of decomposition in a location adjacent to the rookery in Admiralty Bay, King George Island, Antarctica. Of the culturable bacteria, 72 randomly selected colonies were described using 49 morpho-physiological tests, 27 of which were subsequently considered significant in characterizing and differentiating the isolates. On the basis of the nucleotide sequence of a fragment of the 16S rRNA gene in each of 72 pure isolates, three major phylogenetic groups were identified, namely the Moraxellaceae/Pseudomonadaceae (29 isolates), the Flavobacteriaceae (14), and the Micrococcaceae (29). Grouping of the isolates on the basis of morpho-physiological tests (whether 49 or 27 parameters) showed similar results to those based on 16S rRNA gene sequences. Clusters were characterized by considerable intra-cluster variation in both 16S rRNA gene sequences and morpho-physiological responses. High diversity in abundance and morphometry of total bacterial communities during penguin guano decomposition was supported by image analysis of epifluorescence micrographs. The results indicate that the bacterial community in penguin guano is not only one of the richest in Antarctica, but is extremely diverse, both phylogenetically and morpho-physiologically.

The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele. AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp). We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.

The anatomy, morphology and life-cycle of ciliates living on Antarctic krill are described. All ciliates found on the bodies of several thousand individual krill belong to one species of the genus Ephelota. Analysis of small subunit rDNA (SS rDNA) sequences from different Ephelota individuals in various stages of their life-cycle confirmed this result and proved that even the cysts found in great numbers in the filtering baskets of krill represent the resting stage of Ephelota. DNA studies have also shown that Antarctic and non-Antarctic species of Ephelota diverged much earlier than Antarctic and non-Antarctic species of krill.

Background A detailed genetic study of the pre-Columbian population inhabiting the Tompullo 2 archaeological site (department Arequipa, Peru) was undertaken to resolve the kin relationships between individuals buried in six different chullpas. Kin relationships were an important factor shaping the social organization in the pre-Columbian Andean communities, centering on the ayllu, a group of relatives that shared a common land and responsibilities. The aim of this study was to evaluate whether this Andean model of a social organization had an influence on mortuary practices, in particular to determine whether chullpas served as family graves. Results The remains of forty-one individuals were analyzed with both uniparental (mtDNA, Y–chromosome) and biparental (autosomal microsatellites) markers. Reproducible HVRI sequences, autosomal and Y chromosomal STR profiles were obtained for 24, 16 and 11 individuals, respectively. Mitochondrial DNA diversity was comparable to that of ancient and contemporary Andean populations. The Tompullo 2 population exhibited the closest relationship with the modern population from the same region. A kinship analysis revealed complex pattern of relations within and between the graves. However mean relatedness coefficients regarding the pairs of individuals buried in the same grave were significantly higher than those regarding pairs buried in different graves. The Y chromosome profiles of 11 males suggest that only members of one male line were buried in the same grave. Conclusions Genetic investigation of the population that inhabited Tompullo 2 site shows continuity between pre-Columbian and modern Native Amerindian populations inhabiting the Arequipa region. This suggests that no major demographic processes have influenced the mitochondrial DNA diversity of these populations during the past five hundred years. The kinship analysis involving uni- and biparental markers suggests that the community that inhabited the Tompullo 2 site was organized into extended family groups that were buried in different graves. This finding is in congruence with known models of social organization of Andean communities.

International audience; The filamentous fungus Aspergillus nidulans can utilize arginine both as a nitrogen and carbon source. Analysis of areA and areB single and double mutants has shown that the two GATA transcription factors AREA and AREB negatively regulate the expression of arginine catabolism genes agaA and otaA under nitrogen repressing conditions. AREA is necessary for the ammonium repression of agaA and otaA under carbon repressing conditions, while AREB is involved under carbon-limiting conditions. The ability of both AREA and AREB to sense the status of carbon metabolism is most probably dependent on NMRA, and not on the transcription factor CREA, which mediates general carbon catabolite repression in A. nidulans. NMRA is a co-repressor which has previously been shown to bind the C-terminus of AREA and inhibits its activity under conditions of nitrogen sufficiency, in response to high intracellular glutamine levels. We therefore propose a novel function for NMRA, the modulation of AREA and AREB activity in response to the carbon status of the cell.

Expression of Aspergillus nidulans arginine catabolism genes, agaA and otaA, is regulated at the level of transcription by a specific induction and two global carbon and nitrogen repression systems. Post-transcriptional and/or post-translational mechanisms have also been proposed to operate additionally. Gene tagging with transposon impala allowed us to select the rrmA gene. RRMA protein contains three conserved RRM domains, typical for RNA-binding proteins. The gene has a complex structure with several potential transcription start sites, an exceptionally long intron in 5'UTR and few uORFs in the intron. RRMA is highly conserved among fungi. Its homologues, Csx1p of Schizosaccharomyces pombe and Ngr1p of Saccharomyces cerevisiae, participate in the post-transcriptional regulation of specific genes by modifying transcript stability. Levels of otaA and agaA transcripts in the rrmA::impala loss of function mutant grown under inducing conditions are significantly higher than in the wild type strain. We propose that RRMA participates in a mechanism promoting agaA and otaA mRNA degradation. The rrmA::impala mutation has pleiotropic character and results in a slow growth phenotype indicating that rrmA functions are not limited to the regulation of arginine catabolism.

Expression of the arginase structural gene (agaA) in Aspergillus nidulans is subject to complex transcriptional and post-transcriptional regulation. Arginase mRNA has a long 5'-UTR sequence. Analysis of this sequence in silico revealed its putative complex secondary structure, the presence of arginine-binding motifs (arginine aptamers) and a short intron with two potential 3' splicing sites. In this report we present evidence that L-arginine (i) binds directly to the arginase 5'-UTR; (ii) invokes drastic changes in the secondary structure of the 5'-UTR, unlike several other L-amino acids and D-arginine; and (iii) forces the selection of one of two 3' splice sites of an intron present in the 5'-UTR. We postulate that expression of the eukaryotic structural gene coding for arginase in A. nidulans is regulated at the level of mRNA stability, depending on riboswitch-mediated alternative splicing of the 5'-UTR intron.

We have cloned and analysed the arcA gene which encodes a transcriptional activator necessary for the high-level expression of two genes for enzymes of the arginine catabolic pathway in Aspergillus nidulans: agaA (for arginase) and otaA (for ornithine transaminase, OTAse). Here we present complete genomic and cDNA sequences for, and describe the pattern of expression of, the arcA gene. This gene contains one intron and encodes a polypeptide of 600 amino acids. The deduced protein belongs to the family of Zn(2)Cys(6) fungal regulatory proteins. ARCA is the first known protein of this family that has glycine instead of the conserved proline at the fifth position in the second, six-residue, loop of the Zn cluster domain. We have established that transcription of the arcA gene is not self-regulated and does not depend on arginine. Two mutations in arcA, one gain-of-function and one loss-of-function, have been sequenced and the effects of these mutations on the expression of the agaA gene at the transcriptional level are reported.

The arginase structural gene (agaA) from Aspergillus nidulans has been cloned and characterised. Depending on the growth conditions of the mycelium, transcripts of this gene have different 5'ends. These differences could result either from the presence of multiple transcription initiation sites or from differential processing of mRNA. The agaA mRNA has a long 5'UTR with a potentially complex secondary structure. Putative arginine binding aptamers were found in this UTR suggesting interesting possibilities for regulation of the agaA expression.

The ornithine transaminase (otaA) gene of Aspergillus nidulans has been cloned by transformation of the A. nidulans pro – ota – mutant strain with a cosmid gene library. The otaA gene contains two introns and potentially codes for a 453-aa-long protein. The deduced amino-acid sequence is homologous to known ornithine transaminases from Saccharomyces cerevisiae, Plasmodium falciparum, Vigna aconitifolia, rat, mouse and man, particularly in the pyridoxal phosphate-binding domain. The expression of the otaA gene is specifically induced by arginine, and is also under the control of nitrogen-metabolite and carbon-catabolite repression. Regulation of the gene occurs at both transcriptional and post-transcriptional levels. The promoter region of otaA contains putative AREA and CREA binding-sites. Fusion proteins containing AREA or CREA DNA-binding domains bind some of these sites. CREA binding-sites correspond very well to the CREA-binding consensus sequence which is SYGGRG. AREA binding-sites are composed of GATT sequences which are not typical binding sites for the GATA - binding family of transcription factors.

Self-assembly of DNA is considered a fundamental operation in realization of molecular logic circuits. We propose a new approach to implementation of data flow logical operations based on manipulating DNA strands. In our method the logic gates, input, and output signals are represented by DNA molecules. Each logical operation is carried out as soon as the operands are ready. This technique employs standard operations of genetic engineering including radioactive labeling. To check practical utility of the method a series of genetic engineering experiments have been performed. The obtained results confirm interesting properties of the DNA-based molecular data flow logic gates. This technique may be utilized in massively parallel computers.

An Aspergillus nidulans DNA fragment composed of two adjacent Sal I subfragments (1.8 and 0.85 kb) that carries an argB complementing the yeast arg3 mutation has been isolated from two different gene libraries. Hybridization results and immunological tests indicate that the cloned fragment contains the A. nidulans structural gene coding for ornithine carbamoyltransferase (OTCase). Using the cloned gene as a probe, the specific mRNA was identified. The level of this RNA observed in A. nidulans strains grown under various conditions correlated with the level of the OTCase activity, suggesting transcriptional control of OTCase synthesis. Expression of the cloned gene in Saccharomyces cerevisiae does not depend on its orientation in the vector. In Escherichia coli , the cloned gene does not function; however arg - transformants revert to phototrophy with high frequency possibly due to DNA rearrangements within the recombinant plasmid.

Summary: The Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OTCase) is not expressed in Escherichia coli. However, E. coli OTCase-deficient strains transformed with plasmids carrying the argB gene from A. nidulans reverted to prototrophy at a high frequency. In these derivatives the argB gene became functional due to DNA rearrangements upstream of the coding sequence. Two types of rearrangement were characterized. One was identified as an insertion of IS2. The second was a deletion that resulted in transcription of the argB gene from the TcR gene promoter and translation from a newly created ribosome-binding site formed at the junction between the A. nidulans and vector DNA sequences.

The synthesis of two enzymes of the arginine catabolic pathway, arginase and ornithine δ-transaminase (OTAse), in Aspergillus nidulans was found to be sensitive to both glucose and ammonium repression. The glucose and nitrogen starvation result in the identical derepression of OTAse synthesis and have no effects on arginase synthesis. Glucose and ammonium affect the kinetics of induction of both enzymes, however, the effect of ammonium is much stronger. Evidence was obtained for the direct involvement of ammonium in the repression phenomenon. The relations between glucose and ammonium repression are discussed.

Two kinds of mutants of Aspergillus nidulans with altered response of arginine catabolic enzymes to glucose and ammonium repression were obtained. Mutations in the suF locus result in the insensitivity of these enzymes to glucose and to one type of ammonium repression. Mutations in the AniA locus result in hypersensitivity to both types of repression. The enzymes studied can be induced by arginine in AniA mutants only when glucose or the nitrogen source is removed from the medium. The suF mutations are recessive while AniA are dominant. Double suF AniA mutants retain only the suF properties. The functions of both genes and their interrelations are discussed.

In some respiratory deficient cytoplasmic mutants, the buoyant density of mitochondrial DNA is changed to detectable degrees, as compared to that of wild type strain: since this density shift suggests an important modification of polynucleotide sequence in mitochondrial DNA, we examined sequence homology between mitochondrial DNA of the respiratory mutants issued from cytoplasmic or chromosomal mutations. Mitochondrial DNA, nuclear DNA and total RNA were extracted from ϱ+ cells (wild type, respiratory sufficient) and from ϱ- cells (cytoplasmic “petite colonie” mutant, respiratory deficient), and molecular hybridization experiments were carried out between them. When ϱ+ RNA × ϱ+ mitochondrial DNA, formed roughly twice as much hybrids as the heterologous cross, ϱ+ RNA × ϱ1 mitochondrial DNA. Reciprocally, when ϱ- RNA was hybridized to ϱ+ and ϱ- mitochondrial DNA, the homologous cross produced again about twice as much hybrids as the heterologous cross. These results were confirmed by dehydridization-rehybridization experiments: the RNA separated from the hybrids “ϱ+ RNA × ϱ+ mit-DNA” as well as the RNA separated from the hybrids “ϱ+ RNA × ϱ- mit-DNA” were rehybridized either with ϱ+ or ϱ- mit-DNA. A preferential hybridization of ϱ+ RNA with ϱ+ mit-DNA, and of ϱ- RNA with ϱ- mit-DNA was clearly observed. On the contrary, ϱ+ and ϱ- nuclear DNA did not distinguish ϱ+ or ϱ- RNA. The same series of experiments were carried out using a chromosomal mutation,P 7 to p7, leading to the same respiratory deficient phenotype. We found that the p7 mutation did not introduce a detectable change in mitochondrial DNA base sequence. The results support the idea that the cytoplasmic hereditary factor, ϱ, resides in mitochondrial DNA and that the ϱ- mutations studied correspond to a dispersed sequence modification covering about a half of the total mitochondrial DNA genome, leaving the other half unchanged. Alternatively, the results can be explained by a hypothesis in which mitochondrial DNA is a heterogeneous population of the molecules having more or less related sequences and the mutation leads to a selection of certain molecular species. 4 S RNA was found to contain RNA species which hybridize with mitochondrial DNA. The degree of hybridization was very different for ϱ+ and ϱ- S RNA, when they were hybridized with either ϱ+ or ϱ- mitochondrial DNA.

The kaeAKAE1 (suDpro) gene, which was identified in Aspergillus nidulans as a suppressor of proline auxotrophic mutations, encodes the orthologue of Saccharomyces cerevisiae Kae1p, a member of the evolutionarily conserved KEOPS/EKC (Kinase, Endopeptidase and Other Proteins of Small size/Endopeptidase-like and Kinase associated to transcribed Chromatin) complex. In yeast, this complex has been shown to be involved in tRNA modification, transcription, and genome maintenance. In A. nidulans, mutations in kaeA result in several phenotypic effects, the derepression of arginine catabolism genes, and changes in the expression levels of several others, including genes involved in amino acid and siderophore metabolism, sulfate transport, carbon/energy metabolism, translation, and transcription regulation, such as rcoATUP1, which encodes the global transcriptional corepressor.

The transcriptional organization and sequence of the Aspergillus nidulans argB gene, encoding ornithine carbamoyl transferase (OCTase; E.C. 2.1.3.3.), was determined. Transcription of the gene begins within a methionine-initiated open translation reading frame, indicating that a second methionine codon of the open reading frame is used for translation initiation. The predicted length of the OCTase precursor peptide is 359 amino acids, and it contains a highly basic amino terminus that is probably involved in mitochondrial targeting. There is extensive homology between Aspergillus OCTase and mammalian and bacterial OCTases and weaker homology between the Aspergillus polypeptide and bacterial arginine carbamoyl transferase.

Two regulatory mutants for arginine catabolism isolated as proline suppressors were tested for the synthesis of ornithine transcarbamylase (OTC), the arginine anabolic enzyme. Mutations at one locus, suD, result in the insensitivity of OTC synthesis to effectors responsible for the enzyme level in the wild strain. The common genetic regulation of both catabolic and anabolic pathways of arginine is postulated.

The induction of arginase in the fungus Aspergillus nidulans depends on the synthesis of messenger and its subsequent translation, as is shown by selective inhibition of the two processes by proflavine and actidione. In mycelium grown at 30°C messenger synthesis starts with no measurable delay but the enzyme appears 6 min after arginine addition. The stability of the enzyme is accompanied by the lability of its messenger, the coding capacity of which decays with the half-life of 2.7 min. The synthesis of messenger can be separated from its translation by including actidione (5 μg/ml) in an arginine-supplemented culture. After the withdrawal of inducer and inhibitor the expression of arginase messenger takes place. The 4.5-fold increased stability of messenger observed under conditions of blocked translation may be responsible for its accumulation in presence of actidione.

SUMMARY: Proline suppressor mutant su-19 was analysed for arginase and ornithine δ-transaminase (OTA) activity. Both enzymes are constitutively produced by this mutant. Five other suppressor loci not linked with su-19 or to each other are involved in regulation of the two enzymes. A mutant showing no OTA activity was isolated. Properties of this mutant and its effect in various strains of the pro su type were studied.

Comparison of partial mitochondrial 12S and 16S rDNA sequences from non-Antarctic notothenioid fishes – an icefish Champsocephalus esox and two members of the genus Patagonotothen – and their sister species from the Southern Ocean suggests that their divergence took place 1.7 and 6.6–7 million years ago, respectively, i.e. much later than the formation of the Antarctic Polar Front (20–25 million years ago).