The murine MHC encodes a mammalian homolog of bacterial ribosomal protein S13

The mouse major histocompatibility complex (MHC) contains many genes in addition to the classical immune response genes. We have screened overlapping cosmid clones covering 170 kb of the H-2K region for genes expressed in embryonal carcinoma (EC) cells. The Ke-3 gene (Abe et al. 1988) found in this region was further studied by Southern, Northern, and sequence analysis. It is an expressed, intron-containing locus encoding a mouse homolog of the bacterial ribosomal protein S13. This is the first nonorganelle S13 homolog identified in metazoans, and its genomic location has been determined precisely.     

The Drosophila cell survival gene discs lost encodes a cytoplasmic Codanin-1-like protein, not a homolog of tight junction PDZ protein Patj

The Drosophila gene discs lost (dlt) has been reported to encode a homolog of the vertebrate tight junction PDZ protein Patj, and was thought to play a role in cell polarity. Using rescue experiments and sequence analyses, we show that dlt mutations disrupt the Drosophila Codanin-1 homolog, a cytoplasmic protein, and not the PDZ protein. Mutations in human Codanin-1 are associated with congenital dyserythropoietic anemia type I (CDA I). In Drosophila, the genomic organization of dlt is unusual. dlt shares its first untranslated exon with alpha-spectrin, and both genes are coexpressed throughout development. We show that dlt is not required for cell polarity but is needed for cell survival and cell cycle progression. Finally, we present evidence that the PDZ protein previously thought to be encoded by dlt is not required for viability. We propose to rename this PDZ protein after its vertebrate homolog, Patj (Pals-associated tight junction protein).     

NMD3 encodes an essential cytoplasmic protein required for stable 60S ribosomal subunits in Saccharomyces cerevisiae

A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover. Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits. Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes. Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics. However, the mature RNA was rapidly degraded, with a half-life of 4 min. Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients. These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein. Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria. The Nmd3 protein sequence does not contain readily recognizable motifs of known function. However, these proteins all have an amino-terminal domain containing four repeats of Cx2C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization.     

Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation

An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ⁺ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.     

Drosophila spoonbill encodes a dual-specificity A-kinase anchor protein essential for oogenesis

spoonbill is a Drosophila female-sterile mutation, which interferes with normal egg patterning during oogenesis. Previous analyzes linked the mutation to a number of seemingly unrelated pathways, including GRK/EGFR and DPP, two major pathways essential for Drosophila and vertebrate development. Further work suggested that spoonbill may also function in actin polymerization and border-cell migration. Here we describe the molecular cloning of the spoonbill gene and characterize new mutant alleles, further demonstrating that spoonbill's function is essential during oogenesis. We found spoonbill to be allelic to CG3249 (also known as yu), which encodes the only known dual-specificity A-kinase anchor protein in Drosophila. Our data indicate that similar to mammalian AKAPs, Spoonbill protein contains a number of potential kinase and phosphatase binding motifs, and is targeted, in the ovary, to mitochondria and Golgi. Finally, we address some of spoonbill's mutant phenotypes from the perspective of the published data on the AKAP protein family.     

M31, a murine homolog of Drosophila HP1, is concentrated in the XY body during spermatogenesis.

The formation of the sex vesicle, or XY body, during male meiosis and pairing of the sex chromosomes are thought to be essential for successful spermatogenesis. Despite its cytological discovery a century ago, the mechanism of XY body formation, particularly heterochromatinization of the sex chromosomes, has remained unclear. The HP1 class of chromobox genes are thought to encode proteins involved in the packaging of chromosomal DNA into repressive heterochromatin domains, as seen, for example, in position-effect variegation. Study of the distribution of a murine HP1-like chromodomain protein, M31, during spermatogenesis revealed spreading from the tip of the XY body in mid-stage pachytene spermatocytes to include the whole of the XY body in late-pachytene spermatocytes. We also demonstrate that the formation of the XY body during spermatogenic progression in neonatal mice coincides with the expression of a novel nuclear isoform of M31, M31(p21). These results support the view that a common mechanistic basis exists for heterochromatin-induced repression, homeotic gene silencing, and sex-chromosome inactivation during mammalian spermatogenesis.     

The human homolog of the JE gene encodes a monocyte secretory protein.

The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein.     

Spnr, a murine RNA-binding protein that is localized to cytoplasmic microtubules

Previous studies in transgenic mice have established the importance of the 3' untranslated region (UTR) of the spermatid-specific protamine-1 (Prm-1) mRNA in its translational control during male germ cell development. To clone genes that mediate the translational repression or activation of the Prm-1 mRNA, we screened cDNA expression libraries made with RNA from pachytene spermatocytes and round spermatids, with an RNA probe corresponding to the 3' UTR of Prm-1. We obtained six independent clones that encode Spnr, a spermatid perinuclear RNA- binding protein. Spnr is a 71-kD protein that contains two previously described RNA binding domains. The Spnr mRNA is expressed at high levels in the testis, ovary, and brain, and is present in multiple forms in those tissues. Immunolocalization of the Spnr protein within the testis shows that it is expressed exclusively in postmeiotic germ cells and that it is localized to the manchette, a spermatid-specific microtubular array. Although the Spnr protein is expressed too late to be directly involved in the translational repression of Prm-1 specifically, we suggest that the Spnr protein may be involved in other aspects of spermatid RNA metabolism, such as RNA transport or translational activation.     

Nir2, a human homolog of Drosophila melanogaster retinal degeneration B protein,is essential for cytokinesis

Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.     

DOMINO1, a member of a small plant-specific gene family, encodes a protein essential for nuclear and nucleolar functions

Arabidopsis embryos carrying the domino1 mutation grow slowly in comparison with wild type embryos and as a consequence reach only the globular stage at desiccation. The primary defect of the mutation at the cellular level is the large size of the nucleolus that can be observed soon after fertilization in the nuclei of both the embryo and the endosperm. The ultrastructure of mutant nucleoli is drastically different from wild type and points to a fault in ribosome biogenesis. DOMINO1 encodes a protein, which belongs to a plant-specific gene family sharing a common motif of unknown function, present in the tomato DEFECTIVE CHLOROPLASTS AND LEAVES (LeDCL) protein. Using a GFP protein fusion, we show that DOMINO1 is targeted to the nucleus. We propose that inactivation of DOMINO1 has a negative effect on ribosome biogenesis and on the rate of cell division.