Painting of fourth, a chromosome-specific protein in Drosophila

Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila melanogaster, a complex including the five Male-Specific Lethal (MSL) proteins, “paints” the male X chromosome, mediating its hypertranscription. Here, with the molecular cloning of Painting of fourth (Pof), we describe a previously uncharacterized gene encoding a chromosome-specific protein in Drosophila. Unlike the MSL proteins, POF paints an autosome, the fourth chromosome of Drosophila melanogaster. Chromosome translocation analysis shows that the binding depends on an initiation site in the proximal region of chromosome 4 and spreads in cis to involve the entire chromosome. The spreading depends on sequences or structures specific to chromosome 4 and cannot extend to parts of other chromosomes translocated to the fourth. Spreading can also occur in trans to a paired homologue that lacks the initiation region. In the related species Drosophila busckii, POF paints the entire X chromosome exclusively in males, suggesting relationships between the fourth chromosome and the X and between POF complexes and dosage-compensation complexes.

Arthropods: Developmental diversity within a (super) phylum

The expression patterns of developmental genes provide new markers that address the homology of body parts and provide clues as to how body plans have evolved. Such markers support the idea that insect wings evolved from limbs but refute the idea that insect and crustacean jaws are fundamentally different in structure. They also confirm that arthropod tagmosis reflects underlying patterns of Hox gene regulation but they do not yet resolve to what extent Hox expression domains may serve to define segment homologies.

Measuring genome evolution

The determination of complete genome sequences provides us with an opportunity to describe and analyze evolution at the comprehensive level of genomes. Here we compare nine genomes with respect to their protein coding genes at two levels: (i) we compare genomes as “bags of genes” and measure the fraction of orthologs shared between genomes and (ii) we quantify correlations between genes with respect to their relative positions in genomes. Distances between the genomes are related to their divergence times, measured as the number of amino acid substitutions per site in a set of 34 orthologous genes that are shared among all the genomes compared. We establish a hierarchy of rates at which genomes have changed during evolution. Protein sequence identity is the most conserved, followed by the complement of genes within the genome. Next is the degree of conservation of the order of genes, whereas gene regulation appears to evolve at the highest rate. Finally, we show that some genomes are more highly organized than others: they show a higher degree of the clustering of genes that have orthologs in other genomes.

Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context.

Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context. A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon. In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR. Thus, CRP functions either as an activator or as a corepressor. Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription. When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation. Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.

Long-range repression in the Drosophila embryo.

Transcriptional repressors can be characterized by their range of action on promoters and enhancers. Short-range repressors interact over distances of 50-150 bp to inhibit, or quench, either upstream activators or the basal transcription complex. In contrast, long-range repressors act over several kilobases to silence basal promoters. We describe recent progress in characterizing the functional properties of one such long-range element in the Drosophila embryo and discuss the contrasting types of gene regulation that are made possible by short- and long-range repressors.

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.

DNA loops induced by cooperative binding of transcriptional activator proteins and preinitiation complexes.

DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.

Characterization of a Leishmania tropica antigen that detects immune responses in Desert Storm viscerotropic leishmaniasis patients.

A chronic debilitating parasitic infection, viscerotropic leishmaniasis (VTL), has been described in Operation Desert Storm veterans. Diagnosis of this disease, caused by Leishmania tropica, has been difficult due to low or absent specific immune responses in traditional assays. We report the cloning and characterization of two genomic fragments encoding portions of a single 210-kDa L. tropica protein useful for the diagnosis of VTL in U.S. military personnel. The recombinant proteins encoded by these fragments, recombinant (r) Lt-1 and rLt-2, contain a 33-amino acid repeat that reacts with sera from Desert Storm VTL patients and with sera from L. tropica-infected patients with cutaneous leishmaniasis. Antibody reactivities to rLt-1 indicated a bias toward IgG2 in VTL patient sera. Peripheral blood mononuclear cells from VTL patients produced interferon gamma, but not interleukin 4 or 10, in response to rLt-1. No cytokine production was observed in response to parasite lysate. The results indicate that specific leishmanial antigens may be used to detect immune responses in VTL patients with chronic infections.

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

Vector-mediated delivery of a polyamide ("peptide") nucleic acid analogue through the blood-brain barrier in vivo.

Polyamide ("peptide") nucleic acids (PNAs) are molecules with antigene and antisense effects that may prove to be effective neuropharmaceuticals if these molecules are enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier in vivo. The model PNA used in the present studies is an 18-mer that is antisense to the rev gene of human immunodeficiency virus type 1 and is biotinylated at the amino terminus and iodinated at a tyrosine residue near the carboxyl terminus. The biotinylated PNA was linked to a conjugate of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The blood-brain barrier is endowed with high transferrin receptor concentrations, enabling the OX26-SA conjugate to deliver the biotinylated PNA to the brain. Although the brain uptake of the free PNA was negligible following intravenous administration, the brain uptake of the PNA was increased at least 28-fold when the PNA was bound to the OX26-SA vector. The brain uptake of the PNA bound to the OX26-SA vector was 0.1% of the injected dose per gram of brain at 60 min after an intravenous injection, approximating the brain uptake of intravenously injected morphine. The PNA bound to the OX26-SA vector retained the ability to bind to synthetic rev mRNA as shown by RNase protection assays. In summary, the present studies show that while the transport of PNAs across the blood-brain barrier is negligible, delivery of these potential neuropharmaceutical drugs to the brain may be achieved by coupling them to vector-mediated peptide-drug delivery systems.

Physical mapping, cloning, and identification of genes within a 500-kb region containing BRCA1.

BRCA1 is a breast/ovarian cancer susceptibility gene on human chromosome 17q21. We describe a complete and detailed physical map of a 500-kb region of genomic DNA containing the BRCA1 gene and the partial cloning in phage P1 artificial chromosomes. Approximately 70 exons were isolated from this region, 11 of which were components of the BRCA1 gene. Analysis of the other exons revealed a rho-related G protein and the interferon-induced leucine-zipper protein IFP-35.

Análise funcional das proteínas HrcA, GroES/GroEL e DnaK/DnaJ em Caulobacter crescentus

O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem.

Estado sanitário do jacaré-de-papo-amarelo (Caiman latirostris) em paisagens antropizadas no Estado de São Paulo

O jacaré-de-papo (Caiman latirostris) é considerado um crocodiliano de médio porte que apresenta uma ampla distribuição latitudinal na América do Sul. Possivelmente a espécie possui a situação mais complexa entre os crocodilianos brasileiros quanto ao aspecto da conservação, basicamente porque suas populações encontram-se fragmentadas em grande parte de sua área de distribuição original e por utilizarem áreas com fortes atividades antrópicas. Embora a espécie possua aparentemente um processo adaptativo frente a estas pressões, um aspecto fisiológico pode ainda sofrer rápida alteração quando submetido a elas: o estado sanitário. Desta maneira, este estudo objetivou determinar o estado sanitário do maior predador aquático, utilizando duas áreas distintas no Estado de São Paulo, através da determinação dos perfis hematológicos e bioquímicos do sangue, além da caracterização da microbiota oral, servindo de modelo à conservação da espécie em ambientes alterados. No primeiro capítulo foram determinados o perfil hematológico e bioquímico do sangue utilizando-se 29 indivíduos (19 machos e 10 fêmeas) capturados em Angatuba e 11 indivíduos (2 machos, 4 fêmeas e 5 filhotes) capturados em Cubatão. Diferenças estatísticas significativas foram encontradas nos valores de creatinina na comparação entre fêmeas de ambas as áreas de estudo (p = 0,033) e nos valores de alanina aminotransferase (p = 0,003), hemácias (p = 0,034), hemoglobina (p = 0,049) e volume corpuscular médio (p = 0,027) quando da comparação entre sexos. No segundo capítulo determinou-se a microbiota oral destes animais através do isolamento, identificação e caracterização bacteriana, em conjunto com o teste de perfil de susceptibilidade aos antimicrobianos. Foram determinados 14 diferentes tipos de bactérias na área de Angatuba, sendo uma classificada como baciliforme aeróbio Gram-positivo, uma como bacilo Gram-negativo e 12 classificadas como Enterobactérias Gram-negativas. Na área de Cubatão foram isolados cinco tipos de bactérias, sendo quatro classificadas como Enterobactérias Gram-negativas e uma como baciliforme aeróbio Gram-positivo. Considerando que o uso de antimicrobianos é um processo primário no tratamento de animais e pessoas, principalmente nos casos que envolvam infecções provocadas pela interação homem x animais, para ambas as áreas de estudo, as quinolonas Enrofloxacina e Norfloxacina, além do aminoglicosídeo Gentamicina, foram os antimicrobianos que apresentaram os menores índices de resistência frente aos isolados testados.