Mario Zurita

Plasmid vector pBR322 and its special-purpose derivatives: a review

The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmids effectiveness as a cloning vector: plasmid instability in the absence of selection and, the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vectors versatility to accommodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.

Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic.

Fertility is a highly complex and regulated phenomenon essential for the survival of any species. To identify Drosophila fertility‐specific neural networks, we used a GAL4/UAS enhancer trap genetic screen that selectively inactivates groups of neurons. We identified a GAL4 line (bwktqs) that has a female sterile phenotype only when it expresses the tetanus toxin light chain (TeTxLC). These flies lack oviduct contraction, lay almost no eggs, sperm accumulate in the oviducts, and fewer than normal are seen in the storage organs. In insects, two neuroactive substances are important for oviduct contraction: octopamine (OA), a monoamine that inhibits oviduct contraction, and glutamate (Glu), a neurotransmitter that induces contraction. It is known that octopaminergic neurons of the thoracic abdominal ganglion (TAG) modulate oviduct contraction, however, the glutamatergic neurons that innervate the oviduct have not been identified yet and the interaction between these two neuroactive substances is not well understood. Immunostaining experiments revealed that the bwktqs line trapped an octopaminergic neural network that innervates the genital tract. We show that wt like oviduct contraction in TeTxLC‐inactivated flies can only be rescued by simultaneous application of Glu and OA suggesting that the abdominal bwktqs neurons are both octopaminergic and glutamatergic, the use of an agonist and an antagonist for Glu receptors as well as their direct visualization confirmed its participation in this phenomenon. Our work provides the first evidence that adult abdominal type II visceral innervations co‐express Glu and OA and allows us to re‐evaluate the previous model of neuronal network controlling insect oviduct contraction. J. Cell. Physiol. 209: 183–198, 2006.

Open, repair and close again: Chromatin dynamics and the response to UV-induced DNA damage

Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.

The Drosophila trithorax group gene tonalli (tna) interacts genetically with the Brahma remodeling complex and encodes an SP-RING finger protein.

The trithorax group genes are required for positive regulation of homeotic gene function. The trithorax group gene brahma encodes a SWI2/SNF2 family ATPase that is a catalytic subunit of the Brm chromatin-remodeling complex. We identified the tonalli (tna) gene in Drosophila by genetic interactions with brahma. tna mutations suppress Polycomb phenotypes and tna is required for the proper expressions of the Antennapedia, Ultrabithorax and Sex combs reduced homeotic genes. The tna gene encodes at least two proteins, a large isoform (TnaA) and a short isoform (TnaB). The TnaA protein has an SP-RING Zn finger, conserved in proteins from organisms ranging from yeast to human and thought to be involved in the sumoylation of protein substrates. Besides the SP-RING finger, the TnaA protein also has extended homology with other eukaryotic proteins, including human proteins. We show that tna mutations also interact with mutations in additional subunits of the Brm complex, with mutations in subunits of the Mediator complex, and with mutations of the SWI2/SNF2 family ATPase gene kismet. We propose that Tna is involved in postranslational modification of transcription complexes.

Drosophila p53 Is Required to Increase the Levels of the dKDM4B Demethylase after UV-induced DNA Damage to Demethylate Histone H3 Lysine 9

Chromatin undergoes a variety of changes in response to UV-induced DNA damage, including histone acetylation. In human and Drosophila cells, this response is affected by mutations in the tumor suppressor p53. In this work, we report that there is a global decrease in trimethylated Lys-9 in histone H3 (H3K9me3) in salivary gland cells in wild type flies in response to UV irradiation. In contrast, flies with mutations in the Dmp53 gene have reduced basal levels of H3K9me3, which are then increased after UV irradiation. The reduction of H3K9me3 in response to DNA damage occurs preferentially in heterochromatin. Our experiments demonstrate that UV irradiation enhances the levels of Lys-9 demethylase (dKDM4B) transcript and protein in wild type flies, but not in Dmp53 mutant flies. Dmp53 binds to a DNA element in the dKdm4B gene as a response to UV irradiation. Furthermore, heterozygous mutants for the dKdm4B gene are more sensitive to UV irradiation; they are deficient in the removal of cyclobutane-pyrimidine dimers, and the decrease of H3K9me3 levels following DNA damage is not observed in dKdm4B mutant flies. We propose that in response to UV irradiation, Dmp53 enhances the expression of the dKDM4B histone demethylase, which demethylates H3K9me3 preferentially in heterochromatin regions. This mechanism appears to be essential for the proper function of the nucleotide excision repair system.

DNA Repair and Transcriptional Deficiencies Caused by Mutations in the Drosophila p52 Subunit of TFIIH Generate Developmental Defects and Chromosome Fragility

ABSTRACT The transcription and DNA repair factor TFIIH is composed of 10 subunits. Mutations in the XPB, XPD, and p8 subunits are genetically linked to human diseases, including cancer. However, no reports of mutations in other TFIIH subunits have been reported in higher eukaryotes. Here, we analyze at genetic, molecular, and biochemical levels the Drosophila melanogaster p52 (DMP52) subunit of TFIIH. We found that DMP52 is encoded by the gene marionette in Drosophila and that a defective DMP52 produces UV light-sensitive flies and specific phenotypes during development: organisms are smaller than their wild-type siblings and present tumors and chromosomal instability. The human homologue of DMP52 partially rescues some of these phenotypes. Some of the defects observed in the fly caused by mutations in DMP52 generate trichothiodystrophy and cancer-like phenotypes. Biochemical analysis of DMP52 point mutations introduced in human p52 at positions homologous to those of defects in DMP52 destabilize the interaction between p52 and XPB, another TFIIH subunit, thus compromising the assembly of the complex. This study significantly extends the role of p52 in regulating XPB ATPase activity and, consequently, both its transcriptional and nucleotide excision repair functions.

Genes and peptides from the scorpion Centruroides sculpturatus Ewing, that recognize Na(+)-channels.

Sixteen different genes were cloned from the venomous glands of Centruroides sculpturatus Ewing using RNA extracted from scorpions collected in Tucson, Arizona. Based on the amino acid sequence similarities of the proteins coded by these genes, all together there are 22 different structural components in this venom, thought to be specific for Na(+)-channels. The genes reported contain signal peptides with 19 amino acid residues followed by mature peptides of 63-66 amino acid residues in length. One of them correspond to toxin I (CsEI), a known scorpion toxin specific for Na(+)-channels. Four different genes are almost identical to variant 1 (Csv1), presenting only one amino acid change from the original protein. For variant 2 (Csv2) four related genes were found, with only one amino acid change in their primary sequences. Another gene resembles to variant 3 (Csv3, the best known Centruroides sculpturatus toxin), with only three amino acid changes in their primary sequences. Additionally, two genes show variations only on the nucleotide sequence at level of the signal peptides, and several genes clearly show sequences that suggest post-transcriptional modifications, during the maturation process. A phylogenetic tree was generated with the primary structures available and three main divergent branches were found.

Construction and characterization of new cloning vehicles VII. Construction of plasmid pBR327par, a completely sequenced, stable derivatieve of pBR327 containing the par locus of pSC101

In vitro recombinant DNA experiments, using plasmid pBR327 and a DNA fragment derived from plasmid pSC101 containing the par region, resulted in the construction of plasmid pBR327par. This new cloning vehicle has all the cloning properties of the parental plasmid, and is more stable than pBR327. Since the nucleotide sequence of the par region has been determined, this new vector is completely characterized. Some features of the sequence with possible functional significance are discussed.

Antisense suppression of the putative ribosomal protein S3A gene disrupts ovarian development in Drosophila melanogaster

Abstract The Drosophila melanogaster homologue of the Anopheles gambiae C3 cDNA has been isolated and characterized by sequence analysis. The encoded protein was localized by immunochemical and immunocytochemical methods. The Drosophila C3 protein is highly similar to homologues of disputed function, which have previously been identified in fungi, plants and animals. The protein is ubiquitous and localized in the cytoplasm. Cell fractionation followed by detection with a specific antibody preparation shows that the protein is associated with the 40S ribosomal subunit. The C3 gene is located in section 101F of chromosome 4. Antisense transgenic analysis shows that this gene is essential for oogenesis. The most prominent phenotype resulting from antisense depletion of C3 RNA is disappearance of the follicular cells of the ovary (where the concentration of C3 protein is normally high) and abnormalities of the associated germline derivatives, leading to failure of egg production.

Role of the p53 homologue from Drosophila melanogaster in the maintenance of histone H3 acetylation and response to UV-light irradiation

It has been demonstrated that the human tumor suppressor p53 has an important role in modulating histone modifications after UV light irradiation. In this work we explored if the p53 Drosophila homologue has a similar role. Taking advantage of the existence of polytene chromosomes in the salivary glands of third instar larvae, we analyzed K9 and K14 H3 acetylation patterns in situ after UV irradiation of wild‐type and Dmp53 null flies. As in human cells, after UV damage there is an increase in H3 acetylation in wild‐type organisms. In Dmp53 mutant flies, this response is significantly affected at the K9 position. These results are similar to those found in human p53 mutant tumor cells with one interesting difference, only the basal H3 acetylation of K14 is reduced in Dmp53 mutant flies, while the basal H3‐K9 acetylation is not affected. This work shows, that the presence of Dmp53 is necessary to maintain normal H3‐K14 acetylation levels in Drosophila chromatin and that the function of p53 to maintaining histone modifications, is conserved in Drosophila and humans.