The Salmonella enterica serovar Typhi ltrR-ompR-ompC-ompF genes are involved in resistance to the bile salt sodium deoxycholate and in bacterial transformation.

A characterization of the LtrR regulator, an S. Typhi protein belonging to the LysR family is presented. Proteomics, outer membrane protein profiles and transcriptional analyses demonstrated that LtrR is required for the synthesis of OmpR, OmpC and OmpF. DNA-protein interaction analysis showed that LtrR binds to the regulatory region of ompR and then OmpR interacts with the ompC and ompF promoters inducing porin synthesis. LtrR-dependent and independent ompR promoters were identified, and both promoters are involved in the synthesis of OmpR for OmpC and OmpF production. To define the functional role of the ltrR-ompR-ompC-ompF genetic network, mutants in each gene were obtained. We found that ltrR, ompR, ompC and ompF were involved in the control of bacterial transformation, while the two regulators and ompC are necessary for the optimal growth of S. Typhi in the presence of one of the major bile salts found in the gut, sodium deoxycholate. The data presented establish the pivotal role of LtrR in the regulatory network of porin synthesis and reveal new genetic strategies of survival and cellular adaptation to the environment used by Salmonella.

The CRISPR/Cas immune system is an operon regulated by LeuO, H-NS, and leucine-responsive regulatory protein in Salmonella enterica serovar Typhi.

Prokaryotes have developed multiple strategies to survive phage attack and invasive DNA. Recently, a novel genetic program denominated the CRISPR/Cas system was demonstrated to have a role in these biological processes providing genetic immunity. This defense mechanism is widespread in the Archaea and Bacteria, suggesting an ancient origin. In the last few years, progress has been made regarding the functionality of the CRISPR/Cas system; however, many basic aspects of the system remain unknown. For instance, there are few studies about the conditions and regulators involved in its transcriptional control. In this work, we analyzed the transcriptional organization of the CRISPR/Cas system as well as the positive and negative regulators involved in its genetic expression in Salmonella enterica serovar Typhi. The results obtained show that in S. Typhi the CRISPR/Cas system is a LeuO-dependent operon silenced by the global regulator LRP, in addition to the previously known nucleoid-associated protein H-NS; both LRP and H-NS bind upstream and downstream of the transcriptional start site of casA. In this study, relevant nucleotides of the casA regulatory region that mediate its LeuO transcriptional activation were identified. Interestingly, specific growth conditions (N-minimal medium) were found for the LeuO-independent expression of the CRISPR/Cas system in S. Typhi. Thus, our work provides evidence that there are multiple modulators involved in the genetic expression of this immune system in S. Typhi IMSS-1.

Regiones del cromosoma 5 asociadas a características de crecimiento en ganado criollo romosinuano / Chromosomal 5 regions associated with growth traits in the romosinuan o creole cattle

El crecimiento es una de las características de naturaleza cuantitativa que influye en la calidad de la canal. En el presente estudio se identificaron las regiones cromosomales responsables para la variación del crecimiento en el cromosoma 5 en una población de ganado criollo romosinuano. Se evaluaron en 72 progenies las características de peso alnacimiento, peso al destete, peso a los 12 meses y a los 16 meses, área de ojo del lomo alos 12 y 16 meses, espesor de grasa dorsal a los 12 y 16 meses, espesor de grasa del anca a los 12 y 16 meses, ganancia diaria predestete y posdestete, al igual que los genotipos de tres polimorfismos de nucleótido simple de los genes MYF5, PDE1B e IGF1 y decuatro microsatélites BM6026, CSSM34, RM500, ETH10, distribuidos a lo largo del cromosoma. Se realizó un análisis de regresión linear el cual mostró el efecto de seis loci de rasgos cuantitativos (QTL) asociados a características de crecimiento. Cinco QTL fueron significativos (p≤0,05) para las características de peso al nacimiento, peso a los16 meses, área del ojo de lomo a los 12 y 16 meses y ganancia diaria posdestete y un QTL se encontró con una significancia de p≤0,01 para la característica ganancia diaria predestete. Los resultados demostraron que los genes MYF5, PDE1B, IGF1 pueden ser genes candidatos posicionales que inciden en la variación del crecimiento para la calidad de la canal en el ganado romosinuano.

Growth is a quantitative trait that influences the carcass quality. In the present study, bovine chromosomal 5 regions responsible for growth variations have been identified in a Romosinuano creole cattle population. The traits birth weight, weaning weight,12 months weight, 16 months weight, the rib eye area at 12 and 16 months, back fatthickness at 12 and 16 months, rump fat thickness at 12 and 16 months, preweaning and postweaning daily gain were evaluated in 72 progenies, as well as the genotypes of 3 SNPs from the MYF5, PDE1B, IGF1 genes and 4 microsatellites (BM6026,CSSM34, RM500, ETH10) distributed along the chromosome. A linear regression analysis showed the six QTL effect associated with growth traits. Five QTL were significant at p≤0.05 for the triait birth weight, weight at 16 months, rib eye area at 12 and 16 months, and preweaning daily gain interval between 45 and 105 cM and aQTL showed a level significance of p ≤ 0.01 for the postweaning daily gain. It was demostrated according to the position of the QTLs identified in the present study, that the MYF5, PDE1B and IGF1 genes might be positional candidate genes affecting the growth variation for carcass quality in Romosinuano cattle.

Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network.

GMAP-210 (Golgi-microtubule-associated protein of 210 kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83--98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105--196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.

GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein.

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG.

We have isolated a human cDNA clone encoding a novel protein of 22 kDa that is a human counterpart of the rat oncoprotein PTTG. We show that the corresponding gene (hpttg) is overexpressed in Jurkat cells (a human T lymphoma cell line) and in samples from patients with different kinds of hematopoietic malignancies. Analysis of the sequence showed that hPTTG has an amino-terminal basic domain and a carboxyl-terminal acidic domain, and that it is a proline-rich protein with several putative SH3-binding sites. Subcellular fractionation studies show that, although hPTTG is mainly a cytosolic protein, it is partially localized in the nucleus. In addition we demonstrate that the acidic carboxyl-terminal region of hPTTG acts as a transactivation domain when fused to a heterologous DNA binding domain, both in yeast and in mammalian cells.

Networks of transcriptional regulation encoded in a grammatical model.

The work here presented enriches a previous grammatical model of the transcriptional regulation of gene expression. The previous model is centered on the representation of the regulatory regions upstream of genes, and their internal organization in the DNA. This paper is centered in discussing some alternatives related to the representation of the organization of operons and their alternative states of transcription, as active or inactive units of transcription. Transformational rules can be used to describe the binding and unbinding of regulatory proteins, and the associated representations of (ON/OFF) gene expression. The initial representation of a regulated promoter is linked to that of the operon encoding its regulatory protein. In this way the representation of a regulated operon depends on that of all others regulating its transcription, enabling in principle the encoding of regulatory networks within an expanded grammatical model of gene regulation.

Grb2 and its apoptotic isoform Grb3-3 associate with heterogeneous nuclear ribonucleoprotein C, and these interactions are modulated by poly(U) RNA.

Grb2 is an adaptor molecule comprising one Src homology (SH) 2 and two SH3 domains. This protein has a natural isoform named Grb3-3 with a deletion within the SH2 domain. Numerous evidence points to a functional connection between SH2- and SH3-containing proteins and molecules implicated in RNA biogenesis. In this context, we have examined the binding of Grb2 and Grb3-3 to heterogeneous nuclear ribonucleoprotein (hnRNP) C. By the use of an in vivo genetic approach and through in vitro experiments, we furnish evidence that both Grb2 and Grb3-3 interact with hnRNP C proteins. Subcellular fractionation studies clearly show that Grb2 is partially localized in the nucleus. In addition, coimmunoprecipitation experiments demonstrate that Grb2.hnRNP C complexes exist in intact hematopoietic cells. The carboxyl-terminal SH3 domains of Grb2 and Grb3-3 are primarily responsible for the association with hnRNP C. However, although the proline-rich motif of hnRNP C is involved in the interaction with Grb2, it is not in the binding to Grb3-3. Furthermore, poly(U) RNA inhibits the association of Grb2 with hnRNP C, whereas it enhances the interaction between Grb3-3 and hnRNP C. These findings suggest that the Grb2/Grb3-3-hnRNP C interactions might fulfill different biological functions.

NA14 is a novel nuclear autoantigen with a coiled-coil domain.

The serum from a patient with Sjögren's syndrome (RM serum) was used to screen a human testis cDNA expression library. A cDNA of 865 base pairs containing the entire coding sequence for a novel protein was isolated. The 14-kDa predicted protein contains an acidic domain (amino acids 6-80) with a high frequency of heptad repeats characteristic of alpha-helices that form dimeric coiled-coil structures and an alkaline carboxyl-terminal domain (amino acids 81-119). It seems to be widely expressed, but its expression level varies depending on tissues. A protein of apparent molecular mass of 14 kDa was immunoprecipitated from cell lysates by the autoimmune serum, and it was recognized by rabbit antibodies raised to a recombinant bacterial fusion protein generated from the cDNA clone. Conventional and confocal immunofluorescence microscopy on HeLa and 3T3 cells transiently transfected with a tagged form of the protein showed numerous punctate structures scattered throughout the nucleus. This novel protein has been termed NA14 for Nuclear Autoantigen of 14 kDa.

Adenosine deaminase is a specific partner for the Grb2 isoform Grb3-3.

Grb3-3 is an isoform of Grb2, thought to arise by alternative splicing, that lacks a functional SH2 domain but retains functional SH3 domains, which allow interaction with other proteins through binding to prolinerich sequences. Several evidences suggest that besides common partners for Grb2 and Grb3-3, specific targets could exist. In order to find specific partners for Grb3-3, we have screened a human cDNA library by the yeast two-hybrid system with Grb3-3 as a bait. We have identified adenosine deaminase, an enzyme involved in purine metabolism whose deficiency is associated with severe combined immunodeficiency, as a Grb3-3 binding protein that is not able to bind to Grb2. This interaction has been confirmed in vitro with GST fusion proteins and in vivo by coimmunoprecipitation experiments in NIH3T3 cells stably transfected with Grb3-3. The functional significance of this finding is discussed.

A peripheral protein associated with the cis-Golgi network redistributes in the intermediate compartment upon brefeldin A treatment.

Human autoantibodies offer unique tools for the study of cellular constituents since they usually recognize highly conserved components, the most difficult to detect due to their low immunogenicity. The serum from a patient with Sjögren's syndrome (RM serum) showing a very high reactivity to the Golgi complex has been shown to immunoprecipitate and to immunodetect by Western blotting experiments a protein mol wt 210,000 (p210) that was shown to be peripheral and cytoplasmically disposed. A close examination of the p210 labeling revealed some differences with Golgi markers: RM serum staining was slightly more extensive than several Golgi markers and showed a discontinuous or granular appearance. Nocodazole induced a specific and early segregation of many p210-associated vesicles or tubules from Golgi apparatus. Upon brefeldin A treatment, p210 did not redistribute in the ER as did other Golgi proteins. In contrast, it exhibited a vesicular pattern reminiscent to that displayed by proteins residing in the intermediate compartment. Double staining immunofluorescence using the RM serum and the marker of the intermediate compartment, p58, revealed segregation of both proteins in control conditions but colocalization in BFA-treated cells. We have further demonstrated by combining different drug treatments that p210-containing elements in brefeldin A-treated cells belong indeed to the intermediate compartment. Experiments on brefeldin A recovery suggested that these p210 elements might play a role in reformation and repositioning of the Golgi apparatus. Ultrastructural localization performed by immunoperoxidase staining allowed us to establish that p210 interacted with the external side of an abundant tubulo-vesicular system on the cis side of the Golgi complex which extended to connecting structures and vesicles between saccules or stacks of cisternae, p210 appears to be a novel protein residing in the cis-Golgi network that may cycle between the Golgi apparatus and the intermediate compartment.

A high-affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II in the centrosome of human cells.

In the human lymphoblastic cell line KE 37, Northern blot analysis with cDNA probes for human regulatory subunits RII alpha RII beta of the cAMP-dependent protein kinase (A-kinase) type II and immunoblotting or immunoprecipitation studies with several antibodies directed against RII alpha and RII beta show that these two isoforms are expressed. The major isoform alpha is mostly cytosolic, whereas the beta isoform appears concentrated in the Golgi-centrosomal area, as judged by immunofluorescence and cell fractionation. Using a 32P-labelled RII overlay on Western blots, a 350-kDa RII-binding protein (AKAP 350) was specifically identified in centrosomes isolated from this cell line, whereas a Golgi fraction has previously been demonstrated to contain an 85-kDa RII-binding protein (AKAP 85). AKAP 350 is highly insoluble and can partially be extracted from centrosomes as a complex of AKAP 350 and RII subunit. AKAP 350 was identified as a specific centrosomal protein previously demonstrated in the pericentriolar material. The potential significance of a specific subcellular distribution for different RII-binding proteins in nonneuronal cells is discussed.

Production and characterization of three polyclonal antibodies raised against cyst wall proteins of a hypotrichous ciliate.

Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti-p110 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Anti-p52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochemistry. Anti-p110 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti-p66 and anti-p52 antibodies recognized the ectocyst.

Identification of a high affinity binding protein for the regulatory subunit RII beta of cAMP-dependent protein kinase in Golgi enriched membranes of human lymphoblasts.

Immunocytochemical evidence of an association between the regulatory subunit RII of the cAMP-dependent protein kinase (cAMP-PK) and the Golgi apparatus in several cell types has been reported. In order to identify endogenous Golgi proteins binding RII, a fraction enriched in Golgi vesicles was isolated from human lymphoblasts. Only the RII beta isoform was detected in the Golgi-rich fraction, although RII alpha has also been found to be present in these cells. A 85 kDa RII-binding protein was identified in Golgi vesicles using a [32P]RII overlay of Western blots. The existence of an endogenous RII beta-p85 complex in isolated Golgi vesicles was demonstrated by two independent means: (i) co-immunoprecipitation of both proteins under non-denaturing conditions with an antibody against RII beta and (ii) co-purification of RII beta-p85 complexes on a cAMP-analogue affinity column. p85 was phosphorylated by both endogenous and purified catalytic subunits of cAMP-pKII. Extraction experiments and protease protection experiments indicated that p85 is an integral membrane protein although it partitioned atypically during Triton X-114 phase separation. We propose that p85 anchors RII beta to the Golgi apparatus of human lymphoblasts and thereby defines the Golgi substrate targets most accessible to phosphorylation by C subunit. This mechanism may be relevant to the regulation of processes involving the Golgi apparatus itself, such as membrane traffic and secretion, but also relevant to nearby nuclear events dependent on C subunit.