Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication

In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the γ-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication.

whmD is an essential mycobacterial gene required for proper septation and cell division

A study of potential mycobacterial regulatory genes led to the isolation of the Mycobacterium smegmatis whmD gene, which encodes a homologue of WhiB, a Streptomyces coelicolor protein required for sporulation. Unlike its Streptomyces homologue, WhmD is essential in M. smegmatis. The whmD gene could be disrupted only in the presence of a plasmid supplying whmD in trans. A plasmid that allowed chemically regulated expression of the WhmD protein was used to generate a conditional whmD mutant. On withdrawal of the inducer, the conditional whmD mutant exhibited irreversible, filamentous, branched growth with diminished septum formation and aberrant septal placement, whereas WhmD overexpression resulted in growth retardation and hyperseptation. Nucleic acid synthesis and levels of the essential cell division protein FtsZ were unaltered by WhmD deficiency. Together, these phenotypes indicate a role for WhmD in mycobacterial septum formation and cell division.

Essential role for p38α mitogen-activated protein kinase in placental angiogenesis

The p38 family of mitogen-activated protein kinases (MAPKs) mediates signaling in response to environmental stresses and inflammatory cytokines, but the requirements for the p38 MAPK pathway in normal mammalian development have not been elucidated. Here, we show that targeted disruption of the p38α MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. Although chorioallantoic placentation is initiated appropriately in p38α null homozygotes, placental defects are manifest at 10.5 days postcoitum as nearly complete loss of the labyrinth layer and significant reduction of the spongiotrophoblast. In particular, p38α mutant placentas display lack of vascularization of the labyrinth layer as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis. Furthermore, p38α mutants display abnormal angiogenesis in the embryo proper as well as in the visceral yolk sac. Thus, our results indicate a requirement for p38α MAPK in diploid trophoblast development and placental vascularization and suggest a more general role for p38 MAPK signaling in embryonic angiogenesis.

The essential protein encoded by the UL31 gene of herpes simplex virus 1 depends for its stability on the presence of UL34 protein

To pursue an earlier observation that the protein encoded by the UL34 gene binds to intermediate chain of dynein, we constructed a series of mutants from which sequences encoding the entire protein (ΔUL34) or amino-terminal [UL34Δ(3–119)] or carboxyl-terminal [UL34Δ(245–275)] domains were deleted. The mutant lacking the sequence encoding the carboxyl-terminal domain grew in all cell lines tested. The two other mutants replicated only in cell type-dependent manner and poorly. Rescue of ΔUL34 mutant with a fragment that does not encompass the UL31 ORF restored wild-type phenotype. UL34 protein interacts physically with UL31, and the UL31 deletion mutant appears to have a phenotype similar to that of UL34 deletion mutant. Experiments designed to determine whether the phenotypes of the deletion mutants have a common base revealed that cells infected with the ΔUL34 mutant accumulate UL31 RNA but not the corresponding protein. The UL31 protein accumulated, however, to near wild-type virus-infected cell levels in cells infected with ΔUL34 mutant and treated with the MG132 proteosomal inhibitor at 6 h after infection. This is evidence that the stability of an essential viral protein requires the presence of another protein. The observation raises the bar for identification of gene function on the basis of analyses of the phenotype of mutants in which the gene has been deleted or rendered inoperative.

A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1–18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1–18 yeast required the RNA2 5′ noncoding region (NCR), which also conferred a ded1–18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5′ NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1–18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5′ NCRs that appears conserved among bromoviruses. The 5′ attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66 → Gln variant enzyme at 3.1-Å resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

Meiotic lamin C2: The unique amino-terminal hexapeptide GNAEGR is essential for nuclear envelope association

Meiotic lamin C2 is the only A-type lamin expressed during mammalian spermatogenesis. Typical for this short lamin is the unique hexapeptide GNAEGR, which substitutes the nonhelical amino terminus and part of the α-helical rod domain present in somatic lamins. Meiotic lamin C2 also lacks a carboxyl-terminal CaaX box, which is modified by isoprenylation and involved in nuclear envelope (NE) association of somatic isoforms. The mechanism by which lamin C2 becomes localized in the NE is totally unknown. Here we demonstrate that the hexapeptide GNAEGR is essential for this process: (i) Its deletion resulted in a diffuse distribution of lamin C2 within nuclei of transfected COS-7 cells; (ii) Mutated somatic lamin C, containing the sequence GNAEGR at its amino terminus, was located at the NE. The mass spectrometric analysis of the amino terminus of lamin C2 revealed that it is modified by myristoylation. Correspondingly, the substitution of the first glycine residue abolishes the NE association of lamin C2. We conclude that NE association of lamin C2 is achieved by a mechanism different from that of somatic lamins.

PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91phox gene in human neutrophils, monocytes, and B lymphocytes.

Retinoid-related orphan receptor γ (RORγ) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis

To identify the physiological functions of the retinoid-related orphan receptor γ (RORγ), a member of the nuclear receptor superfamily, mice deficient in RORγ function were generated by targeted disruption. RORγ−/− mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that RORγ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of RORγ−/− mice contains 74.4% ± 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of RORγ−/− mice. The latter was supported by the observed increase in annexin V-positive cells. RORγ−/− thymocytes placed in culture exhibit a dramatic increase in the rate of “spontaneous” apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORγ−/− mice. Our observations indicate that RORγ is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which RORγ negatively controls apoptosis in thymocytes.

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte–antigen-presenting cell interface

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav−/− mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav+/− mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav−/− mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: Clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant

A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52/2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription–PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.

RAD50 function is essential for telomere maintenance in Arabidopsis

We have identified and characterized an Arabidopsis thaliana rad50 mutant plant containing a T-DNA insertion in the AtRAD50 gene and showing both meiotic and DNA repair defects. We report here that rad50/rad50 mutant cells show a progressive shortening of telomeric DNA relative to heterozygous rad50/RAD50 controls and that the mutant cell population rapidly enters a crisis, with the majority of the cells dying. Surviving rad50 mutant cells have longer telomeres than wild-type cells, indicating the existence in plants of an alternative RAD50-independent mechanism for telomere maintenance. These results report the role of a protein essential for double-strand break repair in telomere maintenance in higher eukaryotes.

Splicing of constitutive upstream introns is essential for the recognition of intra-exonic suboptimal splice sites in the thrombopoietin gene

The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon–intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.

The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties

Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d1-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d1-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 Å from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd1 nitrite reductase from P. aeruginosa.