The discovery of tetraploidy in the red viscacha rat, Tympanoctomys barrerae (4 n = 102) has emphasized the evolutionary role of genome duplication in mammals. The tetraploid status of this species is corroborated here by in situ PCR and Southern blot analysis of a single-copy gene. The species meiotic configuration strongly suggests a hybrid derivation. To investigate the origin of T. barrerae further, the recently described Pipanacoctomys aureus was studied. This 92-chromosome species also has a duplicated genome size, redundant gene copy number and diploid-like meiotic pairing, consistent with an event of allotetraploidization. Phylogenetic analysis of mitochondrial sequences indicates sister-group relationships between these two tetraploid rodents. The new karyotypic data and the phylogenetic relationships suggest the participation of the ancestral lineages of Octomys mimax in the genesis of P. aureus . The high overall DNA similarity and shared band homology revealed by genomic Southern hybridization as well as matching chromosome numbers between O. mimax and the descendant tetraploid species support the notion of introgressive hybridization between these taxa.

We assessed genome size variation by flow cytometry within and among 31 species of nine families of African and South American hystricognath rodents. Interspecific variation was extensive and genome size was relatively high among the South American radiation whereas only moderate variation and smaller estimates of genome size were observed in the African counterparts. The largest genome size, indicating tetraploidy was recorded in the South American octodontid, Tympanoctomys barrerae (16.8 pg DNA). This quantum shift in DNA content represents a novel mechanism of genome evolution in mammals. As expected in polyploid organisms, varying nucleotypic effects were observed in the dimensions of the sperm cells and lymphocytes of T. barrerae. The role of control mechanisms that influence cell dimensions in polyploid organisms is discussed.

A second Pit-1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon-intron structure was determined, and reverse transcription-polymerase chain reaction (RT-PCR) experiments suggest that only one Pit-1 splice variant is present in carp pituitary. The effect of seasonal acclimatization on the extent of Pit-1 gene expression was studied in summer- and winter-acclimatized carp. Quantitative RT-PCR analysis revealed a clear increase of Pit-1 mRNA in the pituitaries from summer-acclimatized carp compared with the winter-adapted fish. In situ hybridization of pituitary gland sections with riboprobes representing the complete 5'-transactivating region of carp Pit-1 depicted a significantly higher Pit-1 mRNA level in the rostral pars distalis of the summer-acclimatized fish where prolactin is expressed in a manner that resembles the seasonal increase observed in the proximal pars distalis and the pars intermedia. The cell- and temporal-specific transcription of Pit-1 supports its role in the molecular mechanisms that underly the acclimatization process undergone by eurythermal fish as a result of the physical effects of seasonal changes on their habitat.

We isolated a clone comprising four exons of the carp Pit-1 gene. Using synthetic oligonucleotide probes derived from the carp Pit-1 sequence Pit-1 expression was assessed by in situ hybridization in pituitary sections from summer-and winter-acclimatized carp. Semiquantitative analyses of the hybridization signals revealed a significant higher Pit-1 expression in the proximal pars distalis (PPD) and pars intermedia (PI) of the pituitary glands from summeracclimatized carp, compared to the winter-acclimatized fish. In both adaptive states, relative to the PPD and PI, only a basal Pit-1 expression was detected in the rostral pars distalis. Thus, during seasonal acclimatization of an eurythermal fish, Pit-1 seems to be involved in the mechanisms that underlie the compensatory response.

In fish, the innate immune system is the primary response against infection. Toll-like receptors (TLRs) recognize pathogens through pathogen-associated molecular patterns (PAMPs), and some target molecules of TLRs are homologous between fish and mammals. Piscirickettsia salmonis is one of the main pathogens affecting the salmon industry in Chile. Better knowledge of mechanisms underlying its invasive capacity and recognition of target cells is crucial for vaccine development. Therefore, Salmo salar L. TLR1, TLR22, membrane TLR5M and soluble TLR5S sequences were cloned, and expression kinetics were analysed by RT-qPCR in salmon head kidney cells (SHK-1) infected with three different P. salmonis preparations: alive, formaldehyde treated, extract. Clearly, all analysed TLRs were expressed and transcription level changes were revealed at 2 hpi, 12 or 16 hpi and 24 hpi depending on P. salmonis infection scheme. Increased IL1-beta expression confirmed TLR pathway response. Furthermore, significant expression modulations of several members of the TLR pathway in this in vitro model suggest that P. salmonis extract rather than formaldehyde-inactivated bacteria might strengthen the salmon immune system.

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Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human “ubiquitinome” using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport.

Adaptation of eurythermal fish to naturally varying environmental conditions involves modulation of expressions of various factors in the hypothalamo-hypophyseal axis. Here we used three complementary approaches to assess the seasonal variation of growth hormone (GH) protein and mRNA levels in pituitary glands of acclimatized carp fish. First, a polyclonal antibody raised against an oligopeptide derived from the carp GH sequence was used for immunohistochemistry; second, oligonucleotides specific for GH transcripts were used for in situ hybridization. Specific immunodetection of GH coincides with visualization of GH mRNA in the proximal pars distalis, the specific location of somatotroph cells in carp pituitary gland. Finally, competitive RT-PCR analyses confirmed that GH expression exhibits seasonal cyclical reprogramming with higher levels in summer- than in winter-adapted fish. The expression pattern suggests an important role for GH in the molecular mechanisms underlying the acclimatization process. In parallel, amplification of sequences from the fourth intron and adjacent sites from exons IV and V demonstrates the existence of a new GH gene previously undescribed. The detection of transcripts corresponding to each gene suggests that both GH gene copies are active in the duplicated carp genome and that they are similarly affected by seasonal adaptation.