Descripción: This paper reviews basic concepts of modern molecular biology with the premise that its influence in today's medicine is so important that its knowledge cannot remain limited to a few experts. I first analyze the overall structure and organization of human genes, their split nature and the flow of genetic information from DNA to protein. The role of transcriptional control in the regulation of gene expression and cell differentiation is described by introducing experimental examples that define the importance of "master" genes. Basic concepts of genetic engineering, the generation of transgenic and knock out animals and the uses of molecular biology in clinical diagnosis, paternity tests and forensic medicine are presented. Finally, I discuss the possibilities of gene therapy and the fantasies and realities of transgenesis and cloning by nuclear transplant in humans.

Descripción: Many grasshopper species are considered of agronomical importance because they cause damage to pastures and crops. Comprehension of pest population dynamics requires a clear understanding of the genetic diversity and spatial structure of populations. In this study we report on patterns of genetic variation in the South American grasshopper Dichroplus elongatus which is an agricultural pest of crops and forage grasses of great economic significance in Argentina. We use Direct Amplification of Minisatellite Regions (DAMD) and partial sequences of the cytochrome oxydase 1 (COI) mitochondrial gene to investigate intraspecific structure, demographic history and gene flow patterns in twenty Argentinean populations of this species belonging to different geographic and biogeographic regions. DAMD data suggest that, although genetic drift and migration occur within and between populations, measurable relatedness among neighbouring populations declines with distance and dispersal over distances greater than 200 km is not typical, whereas effective gene flow may occur for populations separated by less than 100 km. Landscape analysis was useful to detect genetic discontinuities associated with environmental heterogeneity reflecting the changing agroecosystem. The COI results indicate the existence of strong genetic differentiation between two groups of populations located at both margins of the Paraná River which became separated during climate oscillations of the Middle Pleistocene, suggesting a significant restriction in effective dispersion mediated by females and large scale geographic differentiation. The number of migrants between populations estimated through mitochondrial and DAMD markers suggest that gene flow is low prompting a non-homogeneous spatial structure and justifying the variation through space. Moreover, the genetic analysis of both markers allows us to conclude that males appear to disperse more than females, reducing the chance of the genetic loss associated with recent anthropogenic fragmentation of the D. elongatus studied range. © 2012 Rosetti, Remis.

Descripción: EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ. © 2012 Sala et al.

Descripción: Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ~20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. © 2012 Schor et al.

Descripción: Mucins are highly O-glycosylated molecules which in mammalian cells accomplish essential functions, like cytoprotection and cell-cell interactions. In the protozoan parasite Trypanosoma cruzi, mucin-related glycoproteins have been shown to play a relevant role in the interaction with and invasion of host cells. We have previously reported a family of mucin- like genes in T. cruzi whose overall structure resembled that of mammalian mucin genes. We have now analyzed the relationship between these genes and mucin proteins. A monoclonal antibody specific for a mucin sugar epitope and a polyclonal serum directed to peptide epitopes in a MUC gene-encoded recombinant protein, detected identical bands in three out of seven strains of T. cruzi. Immunoprecipitation experiments confirmed these results. When expressed in eukaryotic cells, the MUC gene product is post-translationally modified, most likely, through extensive O-glycosylation. Gene sequencing showed that the central domains encoding the repeated sequences with the consensus T 8KP 2, varies in number from 1 to 10, and the number of Thr residues in each repeat could be 7, 8, or 10. A run of 16 to 18 Thr residues was present in some, but not all, MUC gene-derived sequences. Direct compositional analysis of mucin core proteins showed that Thr residues are much more frequent than Ser residues. The same fact occurs in MUC gene- derived protein sequences. Molecular mass determinations of the 35-kDa glycoproteins further extend the heterogeneity of the family to the natural mucin molecules. Difficulties in assigning each of the several MUC genes identified to a mucin product arise from the high diversity and partial sequence conservation of the members of this family.

Descripción: RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas. © 2013 Gerez et al.

Descripción: Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNAmediated transcriptional gene silencing. © 2010 Cold Spring Harbor Laboratory Press.

Descripción: Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N. E. Harding, J. M. Cleary, D. K. Cabanas, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tet(r)) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain.

Descripción: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90-95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients. © 2011 Minutolo et al.

Descripción: To understand how the Rhizobium leguminosarum rail-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce rail. Since raiR (and rail) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs. Copyright © 2009, American Society for Microbiology.

Descripción: Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.

Descripción: Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single-particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectory analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained using this novel approach to study chromatin dynamics. © 2008 Springer.

Descripción: The Mediator complex is a greater than 1-megadalton complex, composed of about 30 subunits and found in most eukaryotes, whose main role is to transmit signals from DNA-bound transcription factors to RNA Polymerase II. The proteasome is emerging as an important regulator of transcription during both initiation and elongation. It is increasing the number of cases where the proteolysis of transcriptional activators by the proteasome activates their function. This counterintuitive phenomenon was called "activation by destruction." Here, we show that, in Arabidopsis (Arabidopsis thaliana), PHYTOCHROME AND FLOWERING TIME1 (PFT1), the MEDIATOR25 (MED25) subunit of the plant Mediator complex, is degraded by the proteasome and that proteasome-mediated PFT1 turnover is coupled to its role in stimulating the transcription of FLOWERING LOCUS T, the plant florigen, which is involved in the process of flowering induction. We further identify two novel RING-H2 proteins that target PFT1 for degradation. We show that MED25-BINDING RING-H2 PROTEIN1 (MBR1) and MBR2 bind to PFT1 in yeast (Saccharomyces cerevisiae) and in vitro, and they promote PFT1 degradation in vivo, in a RING-H2- dependent way, typical of E3 ubiquitin ligases. We further show that both MBR1 and MBR2 also promote flowering by PFT1- dependent and -independent mechanisms. Our findings extend the phenomenon of activation by destruction to a Mediator subunit, adding a new mechanism by which Mediator subunits may regulate downstream genes in specific pathways. Furthermore, we show that two novel RING-H2 proteins are involved in the destruction of PFT1, adding new players to this process in plants. © 2012 American Society of Plant Biologists.

Descripción: The section Algarobia of genus Prosopis involves important natural resources in arid and semiarid regions of the world. Their rationale use requires a better knowledge of their biology, genetics and mating system. There are contradictory information about their mating system. Some authors claim they are protogynous and obligate outcrosser. However, some evidence have been shown indicating that they might not be protogynous and that they might be somewhat self-fertile. The current paper analyses genetic structure and mating system parameters in populations of seven species of this section from South and North America based on isozyme data. In all species a significant homozygote excess was found in the offspring population but not in mother plant genotypes. Multilocus and mean single locus outcrossing rates (tm, ts) indicated that about 15 % selfing can occur in the studied populations. The heterogeneity between pollen and ovule allele frequencies was low suggesting population structuration, in agreement with the estimates of correlation of tm within progeny (rt) and correlation of outcrossed paternity (rp). The difference of F(IS) estimates between offspring and mother plants suggest some selection favouring heterozygotes between seedling and adult stages.

Descripción: p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B trans-activation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.

Descripción: Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1Δent2Δ cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity. © 2006 by The National Academy of Sciences of the USA.

Descripción: FKBP51 and FKBP52 (FK506-binding protein 51 and 52) are tetratricopeptide repeat-domain immunophilins belonging to the tetratricopeptide- protein•hsp90•hsp70•p23 heterocomplex bound to steroid receptors. Immunophilins are related to receptor folding, subcellular localization, and hormonedependent transcription. Also, they bind the immunosuppressant macrolide FK506, which shows neuroregenerative and neuroprotective actions by a still unknown mechanism. In this study, we demonstrate that in both, undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the FKBP52• hsp90•p23 heterocomplex concentrates in a perinuclear structure. Upon cell stimulation with FK506, this structure disassembles and this perinuclear area becomes transcriptionally active. The acquisition of a neuronal phenotype is accompanied by increased expression of bIII-tubulin, Map-2, Tau-1, but also hsp90, hsp70, p23, and FKBP52. During the early differentiation steps, the perinuclear heterocomplex redistributes along the cytoplasm and nascent neurites, p23 binds to intermediate filaments and microtubules acquired higher filamentary organization. While FKBP52 moves towards neurites and concentrates in arborization bodies and terminal axons, FKBP51, whose expression remains constant, replaces FKBP52 in the perinuclear structure. Importantly, neurite outgrowth is favored by FKBP52 over-expression or FKBP51 knock-down, and is impaired by FKBP52 knock-down or FKBP51 over-expression, indicating that the balance between these FK506-binding proteins plays a key role during the early mechanism of neuronal differentiation. © 2010 The Authors.

Descripción: The inversion polymorphism of the cactophilic fly Drosophila buzzatii was studied in two natural populations. We assessed the temporal changes and microspatial population structure. We observed a significant increase in the frequency of arrangement 2J at the expense of 2ST in both populations. These gene arrangements appear to affect the life-history of flies differently. Environmental heterogeneity explains the karyotype coexistence in nature. The analysis of population structure showed that differentiation of inversion frequencies among individual breeding sites, the rotting clacodes of Opuntia vulgaris, was highly significant. The karyotypic frequencies did not depart significantly from Hardy-Weinberg expectations, neither in individual rots nor in the total population. These results suggest that the observed population structure can be easily accounted by random genetic drift.

Descripción: Second chromosome inversions and genotypic frequencies at seven allozyme loci were determined in a natural population of the cactophilic species Drosophila buzzatii that uses as breeding sites the necrotic cladodes of the prickly pear Opuntia quimilo and the rotting stems of cardón, Trichocereus terschekii. Different processes govern the evolutionary fate of inversion and allozyme polymorphisms. A pattern of heterotic balance for inversions seems to be acting uniformly in each breeding site and could depend on different regimes of density-dependent selection within cactus hosts. Patterns of variation of allozymes revealed significant heterogeneity in allele frequencies for Esterase-1 (Est-1) among O. quimilo rots and Aldehyde oxidase (Aldox) and Xanthine dehydrogenase (Xdh) among T. terschekii substrates and showed gene-cactus effects only for Esterase-2 (Est-2). Consistent and significant excesses of homozygotes were detected at both the within-rot and in the total population levels that could be accounted for by diversifying selection among individual breeding sites.