The Drosophila simulans Y chromosome interacts with the autosomes to influence male fitness.

The Y chromosome should degenerate because it cannot recombine. However, male-limited transmission increases selection efficiency for male-benefit alleles on the Y, and therefore, Y chromosomes should contribute significantly to variation in male fitness. This means that although the Drosophila Y chromosome is small and gene-poor, Y-linked genes are vital for male fertility in Drosophila melanogaster and the Y chromosome has large male fitness effects. It is unclear whether the same pattern is seen in the closely related Drosophila simulans. We backcrossed Y chromosomes from three geographic locations into five genetic backgrounds and found strong Y and genetic background effects on male fertility. There was a significant Y-background interaction, indicating substantial epistasis between the Y and autosomal genes affecting male fertility. This supports accumulating evidence that interactions between the Y chromosome and the autosomes are key determinants of male fitness.

The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA.

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.

Equine infectious anemia virus gag and pol genes: relatedness to visna and AIDS virus.

Comparison of HTLV-III, the putative AIDS virus, with other related viruses, may help to reveal more about the origin of AIDS in humans. In this study, the nucleotide sequence of the gag and pol genes of an equine infectious anemia virus (EIAV) proviral DNA clone was determined. The sequence was compared with that of HTLV-III and of visna, a pathogenic lentivirus of sheep. The results show that these viruses constitute a family clearly distinct from that of the type C viruses or the BLV-HTLV-I and -II group. Within the family, EIAV, HTLV-III, and visna appear to be equally divergent from a common evolutionary ancestor.

Deletion of a conserved juxtamembrane sequence in Trk abolishes NGF-promoted neuritogenesis.

Deletion of a conserved juxtamembrane sequence (KFG) in the Trk NGF receptor resulted in impaired neurite outgrowth, somatic hypertrophy, and induction of c-fos, c-jun, and TIS1 immediate-early genes. In contrast, these receptors retained the ability to mediate NGF-promoted survival and TIS8 and TIS11 immediate-early gene induction. The mutated receptor also mediated unimpaired autophosphorylation; SHC, PLC-gamma 1, and ERK tyrosine phosphorylation; and PI-3 kinase and ERK activation. However, SNT protein tyrosine phosphorylation, which wild-type receptors mediate via a ras-independent pathway, was undetectable. These findings indicate that the KFG sequence is indispensable for activating a ras-independent NGF signaling pathway involved in promoting neuronal differentiation and highlight potential roles of non-tyrosine-containing receptor domains in growth factor signal transduction.

Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses.

In response to NGF, the Trk receptor tyrosine kinase forms a complex with SHC, a protein that couples receptor tyrosine kinases to p21ras. Complex formation between Trk and SHC, SHC tyrosine phosphorylation, and association of SHC with Grb2 were mediated by autophosphorylation at Y490 in Trk [sequence: see text]. To determine the role of SHC and other Trk substrates in NGF signaling, Trk receptors with mutations in Y490 and Y785 (the PLC-gamma 1 association site) were introduced into PC12nnr5 cells. NGF treatment of PC12nnr5 cells expressing Trk with mutations in either substrate-binding site resulted in normal neurite outgrowth and Erk1 activity and tyrosine phosphorylation. However, PC12nnr5 cells expressing Trk with mutations at both sites failed to stably extend neurites and efficiently induce Erk1 activity and tyrosine phosphorylation in response to NGF. We postulate that Trk receptors can activate Erk1 by either SHC- or PLC-gamma 1-dependent signaling pathways. These results suggest a model whereby Trk receptors utilize at least partially redundant signal transduction pathways to mediate NGF responses.

95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site.

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.

The RESID Database of protein structure modifications and the NRL-3D Sequence-Structure Database.

The RESID Database is a comprehensive collection of annotations and structures for protein post-translational modifications including N-terminal, C-terminal and peptide chain cross-link modifications. The RESID Database includes systematic and frequently observed alternate names, Chemical Abstracts Service registry numbers, atomic formulas and weights, enzyme activities, taxonomic range, keywords, literature citations with database cross-references, structural diagrams and molecular models. The NRL-3D Sequence-Structure Database is derived from the three-dimensional structure of proteins deposited with the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The NRL-3D Database includes standardized and frequently observed alternate names, sources, keywords, literature citations, experimental conditions and searchable sequences from model coordinates. These databases are freely accessible through the National Cancer Institute-Frederick Advanced Biomedical Computing Center at these web sites: http://www. ncifcrf.gov/RESID, http://www.ncifcrf.gov/NRL-3D; or at these National Biomedical Research Foundation Protein Information Resource web sites: http://pir.georgetown.edu/pirwww/dbinfo/resid .html, http://pir.georgetown.edu/pirwww/dbinfo/nrl3d .html

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Features of spliceosome evolution and function inferred from an analysis of the information at human splice sites.

An information analysis of the 5' (donor) and 3' (acceptor) sequences spanning the ends of nearly 1800 human introns has provided evidence for structural features of splice sites that bear upon spliceosome evolution and function: (1) 82% of the sequence information (i.e. sequence conservation) at donor junctions and 97% of the sequence information at acceptor junctions is confined to the introns, allowing codon choices throughout exons to be largely unrestricted. The distribution of information at intron-exon junctions is also described in detail and compared with footprints. (2) Acceptor sites are found to possess enough information to be located in the transcribed portion of the human genome, whereas donor sites possess about one bit less than the information needed to locate them independently. This difference suggests that acceptor sites are located first in humans and, having been located, reduce by a factor of two the number of alternative sites available as donors. Direct experimental evidence exists to support this conclusion. (3) The sequences of donor and acceptor splice sites exhibit a striking similarity. This suggests that the two junctions derive from a common ancestor and that during evolution the information of both sites shifted onto the intron. If so, the protein and RNA components that are found in contemporary spliceosomes, and which are responsible for recognizing donor and acceptor sequences, should also be related. This conclusion is supported by the common structures found in different parts of the spliceosome.

The role of the NPXY motif in the insulin receptor in tyrosine phosphorylation of insulin receptor substrate-1 and Shc.

The insulin receptor phosphorylates insulin receptor substrate-1 (IRS-1) and Shc on tyrosine residues, both of which associate with the protein-abundant Src homology/growth factor receptor-bound protein 2(ASH/GRB2) leading to p21ras activation. Juxtamembrane Tyr960 of the insulin receptor required for tyrosine phosphorylation of both IRS-1 and Shc is contained in the NPXY motif, which is also present in other tyrosine kinase receptors and oncogene products. In this study, the role of this motif in insulin's signaling was examined in Chinese hamster ovary cells expressing insulin receptors with mutations in this motif. All alterations in Tyr960 examined decreased tyrosine phosphorylation of both IRS-1 and Shc to a similar extent. The replacements of Asn957 and the deletion of NPE impaired tyrosine phosphorylation of Shc and IRS-1, although tyrosine phosphorylation of Shc was more severely affected than that of IRS-1. The amount of ASH/GRB2 bound to IRS-1 and Shc in vitro and in vivo was also decreased in these cells. These data suggest that the NPXY motif in the insulin receptor is important for tyrosine phosphorylation of both IRS-1 and Shc as well as subsequent signaling.

MiR-1 and miR-200 inhibit EMT via Slug-dependent and tumorigenesis via Slug-independent mechanisms.

Epithelial-mesenchymal transition (EMT) is a developmental program of signaling pathways that determine commitment to epithelial and mesenchymal phenotypes. In the prostate, EMT processes have been implicated in benign prostatic hyperplasia and prostate cancer progression. In a model of Pten- and TP53-null prostate adenocarcinoma that progresses via transforming growth factor ß-induced EMT, mesenchymal transformation is characterized by plasticity, leading to various mesenchymal lineages and the production of bone. Here we show that SLUG is a major regulator of mesenchymal differentiation. As microRNAs (miRs) are pleiotropic regulators of differentiation and tumorigenesis, we evaluated miR expression associated with tumorigenesis and EMT. Mir-1 and miR-200 were reduced with progression of prostate adenocarcinoma, and we identify Slug as one of the phylogenetically conserved targets of these miRs. We demonstrate that SLUG is a direct repressor of miR-1 and miR-200 transcription. Thus, SLUG and miR-1/miR-200 act in a self-reinforcing regulatory loop, leading to amplification of EMT. Depletion of Slug inhibited EMT during tumorigenesis, whereas forced expression of miR-1 or miR-200 inhibited both EMT and tumorigenesis in human and mouse model systems. Various miR targets were analyzed, and our findings suggest that miR-1 has roles in regulating EMT and mesenchymal differentiation through Slug and functions in tumor-suppressive programs by regulating additional targets.

MicroRNA-106b-25 cluster expression is associated with early disease recurrence and targets caspase-7 and focal adhesion in human prostate cancer.

The miR-106b-25 microRNA (miRNA) cluster is a candidate oncogene in human prostate cancer. Here, we report that miRNAs encoded by miR-106b-25 are upregulated in both primary tumors and distant metastasis. Moreover, increased tumor miR-106b expression was associated with disease recurrence and the combination of high miR-106b and low CASP7 (caspase-7) expressions in primary tumors was an independent predictor of early disease recurrence (adjusted hazard ratio=4.1; 95% confidence interval: 1.6-12.3). To identify yet unknown oncogenic functions of miR-106b, we overexpressed it in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that CASP7 is a direct target of miR-106b, which was confirmed by western blot analysis and a 3'-untranslated region reporter assay. Moreover, selected phenotypes induced by miR-106b knockdown in DU145 human prostate cancer cells did not develop when both miR-106b and CASP7 expression were inhibited. Further analyses showed that CASP7 is downregulated in primary prostate tumors and metastatic lesions across multiple data sets and is by itself associated with disease recurrence and disease-specific survival. Using bioinformatics, we also observed that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally examined using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and disease outcome and may do so by altering apoptosis- and focal adhesion-related pathways.

Searching for non-B DNA-forming motifs using nBMST (non-B DNA motif search tool).

This unit describes basic protocols on using the non-B DNA Motif Search Tool (nBMST) to search for sequence motifs predicted to form alternative DNA conformations that differ from the canonical right-handed Watson-Crick double-helix, collectively known as non-B DNA, and on using the associated PolyBrowse, a GBrowse-based genomic browser. The nBMST is a Web-based resource that allows users to submit one or more DNA sequences to search for inverted repeats (cruciform DNA), mirror repeats (triplex DNA), direct/tandem repeats (slipped/hairpin structures), G4 motifs (tetraplex, G-quadruplex DNA), alternating purine-pyrimidine tracts (left-handed Z-DNA), and A-phased repeats (static bending). The nBMST is versatile, simple to use, does not require bioinformatics skills, and can be applied to any type of DNA sequences, including viral and bacterial genomes, up to an aggregate of 20 megabasepairs (Mbp).

Sequence logos: a new way to display consensus sequences.

A graphical method is presented for displaying the patterns in a set of aligned sequences. The characters representing the sequence are stacked on top of each other for each position in the aligned sequences. The height of each letter is made proportional to its frequency, and the letters are sorted so the most common one is on top. The height of the entire stack is then adjusted to signify the information content of the sequences at that position. From these 'sequence logos', one can determine not only the consensus sequence but also the relative frequency of bases and the information content (measured in bits) at every position in a site or sequence. The logo displays both significant residues and subtle sequence patterns.

Neurotrophin signal transduction by the Trk receptor.

The initial event in the neuronal differentiation of PC12 cells is the binding of the neurotrophin nerve growth factor (NGF) to the Trk receptor. This interaction stimulates the intrinsic tyrosine kinase activity of Trk, initiating a signalling cascade involving the phosphorylation of intracellular proteins on tyrosine, serine, and threonine residues. These signals are then in turn propagated to other messengers, ultimately leading to differentiation, neurotrophin-dependent survival, and the loss of proliferative capacity. To transmit NGF signals, NGF-activated Trk rapidly associates with the cytoplasmic proteins, SHC, PI-3 kinase, and PLC-gamma 1. These proteins are involved in stimulating the formation of various second messenger molecules and activating the Ras signal transduction pathway. Studies with Trk mutants indicate that the activation of the Ras pathway is necessary for complete differentiation of PC12-derived cells and for the maintenance of the differentiated phenotype. Trk also induces the tyrosine phosphorylation of SNT, a specific target of neurotrophic factor activity in neuronal cells. This review will discuss the potential roles of Trk and the proteins of the Trk signalling pathways in NGF function, and summarize our attempts to understand the mechanisms used by Trk to generate the many phenotypic responses of PC12 cells to NGF.

Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins.

We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells.