A poly-zinc finger protein, designated PZFP1 was identified in
Trypanosoma cruzi for the first time. The protein has 191 amino acids, contains seven motifs Cys(X)
2Cys(X)
4His(X)
4Cys. A recombinant ...PZFP1 was generated in
E. coli and the expected 21
kDa polypeptide co-purified with two other inducible products of about 42 and 63
kDa. Western blot analysis of cell extracts using an anti-PZFP1 antibody recognized a major band of 41
kDa. Electrophoretic mobility shift analysis demonstrated that both, recombinant and native PZFP1, specifically interact with single-stranded DNA or RNA oligonucleotides carrying recognition sequences of other CCHC proteins. The protein was localized mainly in the cytoplasm and nucleus as observed by indirect immunofluorescence analysis. PZFP1 interacted specifically with a
T. cruzi serine–arginine-rich protein (TcSR) in a yeast two-hybrid assay, suggesting a role in pre-mRNA processing.
Pattern formation along the anterior-posterior axis of the Drosophila embryo is organized by asymmetrically distributed maternal transcription factors. They initiate a cascade of spatially restricted ...and interacting zygotic gene activities that provide a molecular blueprint of the larval body at blasioderm stage. The key players in the pattern forming process have been identified. Recent progress has begun to reveal the mechanisms by which coherent positional information of maternal origin becomes transferred into serially repeated zygotic gene expression domains reflecting the metameric body plan of the larva.
The Drosophila Bicoid (Bcd) protein plays a dual role as a transcription and translation factor dependent on the unique DNA and RNA binding properties of the homeodomain (HD). We have used circular ...dichroism and fluorescence spectroscopy to probe the structure and stability of the Bcd-HD, for which a high resolution structure is not yet available. The fluorescence from the single tryptophan residue in the HD (Trp-48) is strongly quenched in the native state but is dramatically enhanced (∼20-fold) upon denaturation. Similar results were obtained with the Ultrabithorax HD (Ubx-HD), suggesting that the unusual tryptophan fluorescence may be a general phenomenon of HD proteins. We have used site-directed mutagenesis to explore the role of aromatic acids in the structure of the Bcd-HD and to evaluate the proposal that interactions between the strictly conserved Trp residue in HDs and nearby aromatic residues are responsible for the fluorescence quenching in the native state. We determined that both Trp-48 and Phe-8 in the N-terminal region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor coordinating the orientation of the N-terminal helix I and the recognition helix for efficient binding to a DNA target.
The life of an mRNA from transcription to degradation offers multiple control check points that regulate gene expression. Transcription, splicing, and translation have been widely studied for many ...years; however, in recent years, new layers of posttranscriptional and posttranslational control have been uncovered. They involve the regulation of the metabolism of mRNA in cytoplasmic foci. They are collections of ribonucleoprotein complexes that, in most cases, remain still uncharacterized, except the processing bodies (PBs) and stress granules (SGs), which have been studied (and reviewed) in detail. A challenging prospective is to know how many different classes of foci exist, which functions they support, how are they formed, and how do they relate one to each other. Here, we present an update of the component of the different granules, a possible function, and hypothesis on their in vivo dynamics related to translational control.
Eukaryotic initiation factor (eIF) 4B is part of the protein complex involved in the recognition and binding of mRNA to the ribosome. DrosophilaeIF4B is a single‐copy gene that encodes two isoforms, ...termed eIF4B‐L (52.2 kDa) and eIF4B‐S (44.2 kDa), generated as a result of the alternative recognition of two polyadeynlation signals during transcription termination and subsequent alternative splicing of the two pre‐mRNAs. Both eIF4B mRNAs and proteins are expressed during the entire embryogenesis and life cycle. The proteins are cytoplasmic with polarized distribution. The two isoforms bind RNA with the same affinity. eIF4B‐L and eIF4B‐S preferentially enhance cap‐dependent over IRES‐dependent translation initiation in a Drosophila cell‐free translation system. RNA interference experiments suggest that eIF4B is required for cell survival, although only a modest reduction in rate of protein synthesis is observed. Overexpression of eIF4B in Drosophila cells in culture and in developing eye imaginal discs promotes cell proliferation.
The anterior determinant bicoid (bcd) of Drosophila is a homeodomain protein. It forms an anterior-to-posterior gradient in the embryo and activates, in a concentration-dependent manner, several ...zygotic segmentation genes during blastoderm formation. Its posterior counterpart, the homeodomain transcription factor caudal (cad), forms a concentration gradient in the opposite direction, emanating from evenly distributed messenger RNA in the egg. In embryos lacking bcd activity as a result of mutation, the cad gradient fails to form and cad becomes evenly distributed throughout the embryo. This suggests that bcd may act in the region-specific control of cad mRNA translation. Here we report that bcd binds through its homeodomain to cad mRNA in vitro, and exerts translational control through a bcd-binding region of cad mRNA.
The
Drosophila genome-sequencing project has revealed a total of seven genes encoding eight eukaryotic initiation factor 4E (eIF4E) isoforms. Four of them (
eIF4E-1,2,
eIF4E-3, eIF4E-4 and
eIF4E-5) ...share exon/intron structure in their carboxy-terminal part and form a cluster in the genome. All eIF4E isoforms bind to the cap (m
7GpppN) structure. All of them, except eIF4E-6 and eIF4E-8 were able to interact with
Drosophila eIF4G or eIF4E-binding protein (4E-BP). eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 rescued a yeast eIF4E-deficient mutant in vivo. Only eIF4E-1 mRNAs and, at a significantly lower level, eIF4E3 and eIF4E-8 are expressed in embryos and throughout the life cycle of the fly. The transcripts of the remaining isoforms were detected from the third instar larvae onwards. This indicates the cap-binding activity relies mostly on eIF4E-1 during embryogenesis. This agrees with the proteomic analysis of the eIF4F complex purified from embryos and with the rescue of
l(3)67Af, an embryonic lethal mutant for the
eIF4E-1,2 gene, by transgenic expression of eIF4E-1. Overexpression of eIF4E-1 in wild-type embryos and eye imaginal discs results in phenotypic defects in a dose-dependent manner.
The floating head (flh) gene in zebrafish encodes a homeodomain protein, which is essential for notochord formation along the entire body axis. flh orthologs, termed Not genes, have been isolated ...from chick and Xenopus, but no mammalian ortholog has yet been identified. Truncate (tc) is an autosomal recessive mutation in mouse that specifically disrupts the development of the caudal notochord. Here, we demonstrate that truncate arose by a mutation in the mouse Not gene. The truncate allele (Nottc) contains a point mutation in the homeobox of Not that changes a conserved Phenylalanine residue in helix 1 to a Cysteine (F20C), and significantly destabilizes the homeodomain. Reversion of F20C in one allele of homozygous tc embryonic stem (ES) cells is sufficient to restore normal notochord formation in completely ES cell-derived embryos. We have generated a targeted mutation of Not by replacing most of the Not coding sequence, including the homeobox with the eGFP gene. The phenotype of NoteGFP/eGFP, NoteGFP/tc, and Nottc/tc embryos is very similar but slightly more severe in NoteGFP/eGFP than in Nottc/tc embryos. This confirms allelism of truncate and Not, and indicates that tc is not a complete null allele. Not expression is abolished in Foxa2 and T mutant embryos, suggesting that Not acts downstream of both genes during notochord development. This is in contrast to zebrafish embryos, in which flh interacts with ntl (zebrafish T) in a regulatory loop and is essential for development of the entire notochord, and suggests that different genetic control circuits act in different vertebrate species during notochord formation.
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