Gabriel Gutiérrez

Impaired bone development and increased mesenchymal progenitor cells in calvaria of RB1−/− mice

We have previously shown that the retinoblastoma protein (pRb) can activate expression of Runx2-dependent, bone-specific genes in cultured cells. We now show that pRb also plays a role early in osteogenesis, and that in primary RB1−/− calvarial cells there is an increased osteoprogenitor pool. To understand pRbs function in vivo, we generated a conditional RB1-KO mouse in which pRb expression is efficiently extinguished in osteoblasts. These animals display an apparent developmental defect in bones, most strikingly in the calvaria. Cultured RB1−/− calvarial osteoblasts fail to cease proliferation upon reaching confluence or following differentiation. Re-plating assays of primary RB1−/− calvarial cells after differentiation showed a clear adipogenic ability with increased multipotency. RB1−/− osteoblasts display a severe reduction in levels of mRNAs expressed late in differentiation. In this study, we present strong evidence that pRb has multiple regulatory roles in osteogenesis. Furthermore, in the absence of RB1−/− there is a larger pool of multipotent cells compared with the WT counterpart. This increased pool of osteoprogenitor cells may be susceptible to additional transforming events leading to osteosarcoma, and is therefore key to understanding RB1 as a target in malignancy.

Preference for Guanosine at First Codon Position in Highly Expressed Escherichia Coli Genes. A Relationship with Translational Efficiency

The variation in base composition at the three codon sites in relation to gene expressivity, the latter estimated by the Codon Adaptation Index, has been studied in a sample of 1371 Escherichia coli genes. Correlation and regression analyses show that increasing expression levels are accompanied by higher frequencies of base G at first, of base A at second and of base C at third codon positions. However, correlation between expressivity and base compositional biases at each codon site was only significant and positive at first codon position. The preference for G-starting codons as gene expression level increases is discussed in terms of translational optimization.

Evolution of the lipocalin family as inferred from a protein sequence phylogeny.

The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles. It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses. We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family. Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency.

Comparative gene expression profile of mouse carotid body and adrenal medulla under physiological hypoxia

The carotid body (CB) is an arterial chemoreceptor, bearing specialized type I cells that respond to hypoxia by closing specific K+ channels and releasing neurotransmitters to activate sensory axons. Despite having detailed information on the electrical and neurochemical changes triggered by hypoxia in CB, the knowledge of the molecular components involved in the signalling cascade of the hypoxic response is fragmentary. This study analyses the mouse CB transcriptional changes in response to low PO2 by hybridization to oligonucleotide microarrays. The transcripts were obtained from whole CBs after mice were exposed to either normoxia (21% O2), or physiological hypoxia (10% O2) for 24 h. The CB transcriptional profiles obtained under these environmental conditions were subtracted from the profile of control non‐chemoreceptor adrenal medulla extracted from the same animals. Given the common developmental origin of these two organs, they share many properties but differ specifically in their response to O2. Our analysis revealed 751 probe sets regulated specifically in CB under hypoxia (388 up‐regulated and 363 down‐regulated). These results were corroborated by assessing the transcriptional changes of selected genes under physiological hypoxia with quantitative RT‐PCR. Our microarray experiments revealed a number of CB‐expressed genes (e.g. TH, ferritin and triosephosphate isomerase) that were known to change their expression under hypoxia. However, we also found novel genes that consistently changed their expression under physiological hypoxia. Among them, a group of ion channels show specific regulation in CB: the potassium channels Kir6.1 and Kcnn4 are up‐regulated, while the modulatory subunit Kcnab1 is down‐regulated by low PO2 levels.

Patterns of Group I Intron Presence in Nuclear SSU rDNA of the Lichen Family Parmeliaceae

Group I introns are commonly reported within nuclear SSU ribosomal DNA of eukaryotic micro-organisms, especially in lichen-forming fungi. We have studied the primary and secondary structure of 70 new nuclear SSU rDNA group I introns of Parmeliaceae (Ascomycota: Lecanorales) and compared them with those available in databases, covering more than 60 species. The analyzed samples of Parmeliaceae fell into two groups, one having an intron at the 1506 site and another lacking this one but having another at the 1516 or 1521 position. Introns at the 1521 position seem to be transposed from 1516 sites. Introns at the 1516 position were similar in structure to ones previously reported at this site and known from other lecanoralean fungi, while those at the 1506 position showed structural differences and no similar introns are known from related fungi. The study of the distribution of group I introns within a large monophyletic ensemble of fungi has revealed an unexpected correlation between intron types and ecological and geographical parameters. The introns at the 1516 position occurred in mainly arctic, boreal, and temperate lichens, while those at position 1506 were present in mainly tropical and subtropical to oceanic mild-temperate taxa. Further, the 1516 introns occurred in genera with few distributed species that could represent older taxa, while the 1506 ones were mainly in species-rich genera that could be of recent speciation, as many species have wide distribution areas. The transition between two different environments has been accompanied by a change in introns gained and lost.

MicroRNA-dependent regulation of transcription in non-small cell lung cancer

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

Relationship between G+C content, ORF-length and mRNA concentration in Saccharomyces cerevisiae

RNA biogenesis is a tightly‐regulated process. The levels and timing of expression of a gene depends on its particular function. However, gene expression levels may also depend on structural features. Here we describe the analysis of gene expression of 4977 ORFs using DNA microarrays covering the whole genome of three different S. cerevisiae strains, wild‐type and tho2 and thp1 mutants with a general effect on mRNA biogenesis. We show that transcripts from G+C‐rich ORFs accumulate at higher concentrations than those from G+C‐poor ones, in different ORF‐length categories in all strains tested. In addition, we found a negative correlation between ORF length and G+C content. Our results indicate that length and G+C content of a gene have a clear effect on its levels of expression. We discuss the biological relevance of these results, as well as different ways that these structural features could modulate mRNA biogenesis. Copyright

Apolipoprotein D alters the early transcriptional response to oxidative stress in the adult cerebellum

J. Neurochem. (2011) 117, 949–960.

A Relationship between Carotenoid Accumulation and the Distribution of Species of the Fungus Neurospora in Spain

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

Significance Photolyases repair UV-B–induced DNA lesions. They form a large protein family together with cryptochrome photoreceptors (cryptochrome/photolyase family, CPF). A more recently discovered CPF subclade consists of DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH), present in bacteria, plants, animals, and fungi. Cry-DASH are considered as photoreceptors with residual repair activity for DNA lesions in single-stranded DNA. Canonical photolyases repair such lesions in single-stranded and double-stranded DNA. Here, we show that mucoromycotina fungi except Umbelopsis ramanniana, which is an early diverging lineage within the mucoralean fungi, encode only cry-DASH. They possess the full spectrum of DNA repair activity as canonical photolyases as exemplified for the Phycomyces CryA. This finding is a unique example of CPF evolution where a canonical CPD-photolyase was lost but its function was maintained by cry-DASH. DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B–induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi.