The 82F late puff contains the L82 gene, an essential member of a novel gene family.

Metamorphosis in Drosophila results from a hierarchy of ecdysone-induced gene expression initiated at the end of the third larval instar. A now classical model of this hierarchy was proposed based on observations of the activity of polytene chromosome "puffs" which distinguished "early" puffs as those directly induced by ecdysone and "late" puffs as those which become active as a secondary response to the hormone. We report here the isolation and characterization of the L82 gene corresponding to the extensively characterized late puff at 82F. L82 is a complex gene that spans at least 50 kb of genomic DNA, produces at least seven different nested mRNAs, and has homology to a novel gene family. In contrast to most previously characterized puff genes, the broad developmental expression pattern of L82 suggests that it is controlled by both ecdysone-dependent and ecdysone-independent regulatory mechanisms. L82 mutations were identified by transgene rescue of developmental delay and eclosion lethal phenotypes.     

Hsl7p, the yeast homologue of human JBP1, is a protein methyltransferase

The yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBP1, a newly characterized protein methyltransferase. In this report, Hsl7p also is shown to be a methyltransferase. It can be crosslinked to [(3)H]S-adenosylmethionine and exhibits in vitro protein methylation activity. Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p, whereas histones H1, H2B, and H3 and bovine cytochrome c were not. We demonstrated that JBP1 can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype, and a point mutation in the JBP1 S-adenosylmethionine consensus binding sequence eliminated all complementation by JBP1. Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBP1. These data provide evidence for an intricate link between protein methylation and macroscopic changes in yeast morphology.     

DIXDC1 isoform, l-DIXDC1, is a novel filamentous actin-binding protein

Ccd1, a DIX domain containing Zebrafish protein involved in neural patterning, is a positive regulator of the Wnt signaling pathway. DIXDC1, the human homolog of Ccd1, has two predominant isoforms. The short form (s-DIXDC1) has a similar amino acid sequence compared with Ccd1, while the long form (l-DIXDC1) contains an extra N-terminal sequence containing a calponin-homology (CH) domain, suggesting additional interaction with actin that we have performed detailed analysis in this report. We show that mRNA expression of both DIXDC1 isoforms can be detected in various adult tissues by Northern blot analysis and is most abundant in cardiac and skeletal muscles. Both endogenous and ectopically expressed l-DIXDC1, but not s-DIXDC1, in cultured mammalian cells is localized to actin stress fibers at the filament ends in focal adhesion plaques. More importantly, l-DIXDC1 can directly bind to filamentous actin both in vitro and in vivo and the binding is mediated via a novel actin-binding domain (ABD) from amino acid 127 to 300. Thus, our data provide the first evidence that l-DIXDC1 may act as a novel branching component in the Wnt signaling pathway targeting both beta-catenin-TCF complex for gene expression and cytoskeleton for regulating dynamics of actin filaments.     

A novel cDNA encoding a human homologue of ribosomal protein L29

During the large scale partial sequencing of human heart cDNA clones, a novel clone which is very similar to the rat ribosomal protein L29 in both DNA and amino acid sequences was found. The cDNA encodes a protein with a deduced molecular weight of 17 751 (159 aa). It shows 80.4% homology to protein L29 from the large ribosomal subunit of rat and is related to yeast YL43. The putative protein was named human ribosomal protein L29 (hRPL29). hRPL29 has a large excess of basic residues over acidic ones. The large amount of charged residues makes the protein very hydrophilic and the protein has a deduced pI of 12.16. Internal repeats have been characterised in many ribosomal proteins and a tandem repeat of KAKAKAKA was found to be unique to hRPL29. Analysis of gene organisation by Southern blotting shows that of the approximate 10 copies of hrpL29, all but one are pseudogenes. Northern analysis indicated that the mRNA that encodes human L29 is approx. 800 base pairs in length. An intron of hrpL29 has also been cloned and sequenced by polymerase chain reaction using human genomic DNA as the template.     

Differential structure and activity between human and mouse intelectin-1: human intelectin-1 is a disulfide-linked trimer, whereas mouse homologue is a monomer

Human intelectin-1 (hITLN-1) is a 120-kDa lectin recognizing galactofuranosyl residues found in cell walls of various microorganisms but not in mammalian tissues. Although mouse intelectin-1 (mITLN-1) has been identified previously, its biochemical properties and functional characteristics have not been studied. Therefore, we have compared structures and saccharide-binding specificities of hITLN-1 and mITLN-1 using recombinant proteins produced by mammalian cells. Recombinant hITLN-1 is a trimer, disulfide-linked through Cys-31 and Cys-48, and N-glycosylated at Asn-163. Despite 84.9% amino acid identity to hITLN-1, recombinant and intestinal mITLN-1 are unglycosylated 30-kDa monomers. Recombinant hITLN-1, as well as recombinant and intestinal mITLN-1 were purified by Ca(2+)-dependent adsorption to galactose-Sepharose. In competitive binding studies, hITLN-1 was eluted from galactose-Sepharose by 100 mM 2-deoxygalactose, a galactofuranosyl disaccharide, d-xylose, and both d- and l-ribose. In contrast, mITLN-1 was partially eluted by the galactofuranosyl disaccharide, and only minimally by the other saccharides indicating that the two intelectins have different saccharide-binding specificities. When the N- and C-terminal regions of hITLN-1 were replaced, respectively, with those of mITLN-1, galactose-Sepharose binding was associated with the C-terminal regions. Finally, hITLN-1 binding to galactose-Sepharose was not affected by the substitution of the Cys residues in the N-terminal region that are necessary for oligomer formation, nor was it affected by the removal of the N-linked oligosaccharide at Asn-163. Although both hITLN-1 and mITLN-1 recognize galactofuranosyl residues, our comparative studies, taken together, demonstrate that these intelectins have different quaternary structures and saccharide-binding specificities.     

hDKIR, a human homologue of the Drosophila kelch protein, involved in a ring-like structure

We have previously purified and cloned an apoptosis-inducing protein (AIP) derived from fish infected with the anisakis simplex. Recently, we identified a series of AIP-responsive genes in the HL-60 cell line using a subtractive hybridization method. Here we report the molecular cloning and characterization of one of these genes, which encodes a novel human kelch protein containing 568 amino acid residues, termed hDKIR. The Drosophila Kelch protein localizes to a ring canal structure, which is required for oocyte development. When hDKIR was expressed in cultured-mammalian cells, hDKIR localized to a ring-like structure. Furthermore, when coexpressed with Mayven or Keap1, hDKIR bound to Mayven and recruited Mayven into ring-like structures perfectly. This indicates that kelch homologues can interact with each other in a specific manner and such interaction can affect the subcellular localization of kelch proteins. Finally, domain analysis revealed that both the N-terminal POZ (poxviruses and zinc fingers) and intervening region (IVR) domains of hDKIR are essential for ring-like structure activity, suggesting that the development of the ring-like structure is independent of the ability to bind actin.     

The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments

ORP1L is a member of the human oxysterol-binding protein (OSBP) family. ORP1L localizes to late endosomes (LEs)/lysosomes, colocalizing with the GTPases Rab7 and Rab9 and lysosome-associated membrane protein-1. We demonstrate that ORP1L interacts physically with Rab7, preferentially with its GTP-bound form, and provide evidence that ORP1L stabilizes GTP-bound Rab7 on LEs/lysosomes. The Rab7-binding determinant is mapped to the ankyrin repeat (ANK) region of ORP1L. The pleckstrin homology domain (PHD) of ORP1L binds phosphoinositides with low affinity and specificity. ORP1L ANK- and ANK+PHD fragments induce perinuclear clustering of LE/lysosomes. This is dependent on an intact microtubule network and a functional dynein/dynactin motor complex. The dominant inhibitory Rab7 mutant T22N reverses the LE clustering, suggesting that the effect is dependent on active Rab7. Transport of fluorescent dextran to LEs is inhibited by overexpression of ORP1L. Overexpression of ORP1L, and in particular the N-terminal fragments of ORP1L, inhibits vacuolation of LE caused by Helicobacter pylori toxin VacA, a process also involving Rab7. The present study demonstrates that ORP1L binds to Rab7, modifies its functional cycle, and can interfere with LE/lysosome organization and endocytic membrane trafficking. This is the first report of a direct connection between the OSBP-related protein family and the Rab GTPases.     

Caenorhabditis elegans ciliary protein NPHP-8, the homologue of human RPGRIP1L, is required for ciliogenesis and chemosensation

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal failure in children and young adults. NPHP8/RPGRIP1L is a novel ciliary gene that, when mutated, in addition to causing NPHP, also causes Joubert syndrome (JBTS) and Meckel syndrome (MKS). The exact function of NPHP8 and how defects in NPHP8 lead to human diseases are poorly understood. Here, we studied the Caenorhabditis elegans homolog nphp-8 (C09G5.8) and explored the possible function of NPHP-8 in ciliated sensory neurons. We determined the gene structure of nphp-8 through rapid amplification of cDNA ends (RACE) analysis and discovered an X-box motif that had been previously overlooked. Moreover, NPHP-8 co-localized with NPHP-4 at the transition zone at the base of cilia. Mutation of nphp-8 led to abnormal dye filling (Dyf) and shorter cilia lengths in a subset of ciliary neurons. In addition, chemotaxis to several volatile attractants was significantly impaired in nphp-8 mutants. Our data suggest that NPHP-8/RPGRIP1L plays an important role in cilia formation and cilia-mediated chemosensation in a cell type-specific manner.     

GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.     

Identification of a novel antiapoptotic human protein kinase C delta isoform, PKCdeltaVIII in NT2 cells

Protein kinase C (PKC) delta plays an important role in cellular proliferation and apoptosis where it is involved in the caspase-3 mediated apoptotic pathway. Cleavage of PKCdeltaI by caspase-3 releases a catalytically active C-terminal fragment that is sufficient to induce apoptosis. In this paper, we identified a novel human PKCdelta isozyme, PKCdeltaVIII (Genbank accession number DQ516383) in human teratocarcinoma (NT2) cells that differentiate into hNT neurons upon retinoic acid (RA) treatment. Expression of PKCdeltaVIII was confirmed by real-time RT-PCR analysis, and we observed that after an initial peak at 24 h following RA treatment, its expression gradually declined with prolonged RA treatment. PKCdeltaVIII is generated via the utilization of an alternative 5' splice site, and this results in an insertion of 31 amino acids in the caspase-3 recognition sequence DMQD. The function of PKCdeltaVIII was examined by subcloning it into an expression vector and raising an antibody specific to PKCdeltaVIII. Using in vivo and in vitro assays, we demonstrated that PKCdeltaVIII is resistant to caspase-3 cleavage. Next, we sought to determine the role of PKCdeltaVIII in apoptosis in NT2 cells. Overexpression of PKCdeltaVIII and knockdown using PKCdeltaVIII siRNA suggest an antiapoptotic function for the PKCdeltaVIII isozyme. We demonstrate that antisense oligonucleotides (ASO) directed toward the 5' splice site I promote the expression of the PKCdeltaVIII isozyme. Our results indicated that ASO mediated PKCdeltaVIII expression rescued NT2 cells from etoposide-induced apoptosis. We conclude that the novel human PKCdeltaVIII splice variant functions as an antiapoptotic protein in NT2 cells.     

Mapping and structure of DMXL1, a human homologue of the DmX gene from Drosophila melanogaster coding for a WD repeat protein

The DmX gene was recently isolated from the X chromosome of Drosophila melanogaster. TBLASTN searches of the dbEST databases revealed sequences with a high level of similarity to DmX in a variety of different species, including insects, nematodes, and mammals showing that DmX is an evolutionarily highly conserved gene. Here we describe the cloning of the cDNA and the chromosomal localization of one of the human homologues of DmX, Dmx-like 1 (DMXL1). The human DMXL1 gene codes for a large mRNA of 11 kb with an open reading frame of 3027 amino acids. The putative protein belongs to the superfamily of WD repeat proteins, which have mostly regulatory functions. The DMXL1 protein contains an exceptionally large number of WD repeat units. The DMXL1 gene is located on chromosome 5q22 as determined by radiation hybrid mapping and fluorescence in situ hybridization. Although the function of the DMXL1 gene and its homologues in other species remains to be discovered, the high level of evolutionary conservation together with the unusual structure suggests that it probably has an important function.