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PubMed2005

Novel rearrangement of chromosome band 22q11.2 causing 22q11 microdeletion syndrome-like phenotype and rhabdoid tumor of the kidney.

The 22q11.2 microdeletion syndrome is the most frequent microdeletion syndrome in humans, yet its genetic basis is complex and is still not fully understood. Most patients harbor a 3-Mb deletion (typically deleted region [TDR]), but occasionally patients with atypical deletions, some of which do not overlap with each other and/or the TDR, have been described. Microduplication of the TDR leads to a phenotype similar, albeit not identical, to the deletion of this region. Here we present a child initially suspected of having 22q11 microdeletion syndrome, who in addition developed a fatal malignant rhabdoid tumor of the kidney. Detailed cytogenetic and molecular analyses revealed a complex de novo rearrangement of band q11 of the paternally derived chromosome 22. This aberration exhibited two novel features. First, a microduplication of the 22q11 TDR was associated with an atypical 22q11 microdeletion immediately telomeric of the duplicated region. Second, this deletion was considerably larger than previously reported atypical 22q11 deletions, spanning 2.8 Mb and extending beyond the SMARCB1/SNF5/INI1 tumor suppressor gene, whose second allele harbored a somatic frameshift-causing sequence alteration in the patient's tumor. Two nonallelic homologous recombination events between low-copy repeats (LCRs) could explain the emergence of this novel and complex mutation associated with the phenotype of 22q11 microdeletion syndrome.

Chromosome Deletion↗

PubMed2003

Studies on the ATP3 gene of Saccharomyces cerevisiae: presence of two closely linked copies, ATP3a and ATP3b, on the right arm of chromosome II.

In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the gamma subunit of the F(1)F(0)-ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single-copy gene on chromosome II. We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F(1)-alpha and F(1)-beta, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F(1)F(0)-ATPase. By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b. The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303-1A, W303-1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a. With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp. The differences between ATP3 with the single base substitution (Ser(308) to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions. However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3. It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild-type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone.

Base Sequence↗

PubMed2000

Formation and stability of the bacteriophage lambda replication complexes in UV-irradiated Escherichia coli.

Bacteriophage lambda replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter lambda DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage lambda, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the lambda replication complex. We found that UV irradiation of the host cells prevented formation of the stable lambda replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable lambda replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of lambda replication complexes is possible. Both stable and unstable lambda replication complexes are functional because replication of lambda DNA under conditions preventing formation of the stable complex proceeds efficiently.

Bacteriolysis↗

PubMed1999

Definition of a high affinity growth hormone DNA response element.

Rat liver contains a growth hormone inducible nuclear factor complex, GHINF, that binds to the growth hormone response element (GHRE) of the serine protease inhibitor (Spi) 2.1 gene. GHINF contains Stat5 and binds to paired gamma-activated sites (GAS) within the GHRE, but poorly to either one alone. By analysis of the sequence of various GAS sites that bind the GHINF complex (based on the GHRE 3' GAS motif), we demonstrate that a 13 nucleotide high affinity DNA recognition sequence (haGHRE) for GHINF complex binding is (ANTTC)C/T(N)A/G(GAA)A/T(A)/T. One copy of the haGHRE will replace the requirement for two GAS elements present in the wild type promoter in supporting a GH response in primary hepatocyte culture. Mutation of the native Spi 2.1 from a paired GAS site to a single haGHRE does not appreciably change its affinity for binding to the GHINF complex, nor does it alter its sensitivity to GH concentration.

Animals↗

PubMed2006

Complex T cell memory repertoires participate in recall responses at extremes of antigenic load.

The CD8 T cell memory response to the HLA-A2-restricted influenza epitope M1(58-66) can be an instructive model of immune memory to a nonevolving epitope of a frequently encountered pathogen that undergoes clearance. This memory repertoire can be complex, composed of a large number of clonotypes represented at low copy numbers, while maintaining a focus on the use of VB17 T cell receptors with identified Ag recognition motifs. Such a repertoire structure might provide a panoply of clonotypes whose differential avidity for the epitope would allow responses under varying antigenic loads. This possibility was tested experimentally by characterizing the responding repertoire in vitro while varying influenza Ag concentration over five orders of magnitude. At higher and lower Ag concentrations there was increased cell death, yet a focused but diverse response could still be observed. Thus, one of the characteristics of complex memory repertoires is to provide effector function at extremes of Ag load, a characteristic that is not generally considered in vaccination development but may be important in measuring its efficacy.

Antigens, Viral↗

PubMedDate not supplied

Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata.

Biological nitrogen fixation requires the nitrogenase enzyme complex, ATP, and a strong reductant. Klebsiella pneumoniae contains 15 linked nitrogen fixation (nif) genes, three of which, nifH, nifD and nifK have been sufficiently conserved in evolution that cloned K. pneumoniae nifHDK DNA will hybridize to DNA sequences from every nitrogen-fixing bacterium examined to date, including the purple, non-sulphur bacterium Rhodopseudomonas capsulata, in which one complete nifHDK operon has been mapped. Using cloned K. pneumoniae nifHDK DNA we report here that R. capsulata contains multiple copies of the genes for nitrogenase components. Two regions containing sequences homologous to all three nif structural genes have been identified, and mutations in one region produced a Nif- phenotype. Nif+ pseudorevertants were derived from these mutants, some of which retained the original mutation suggesting that some of the extra nif gene sequences can be functionally activated.

Chromosome Deletion↗

PubMed1991

The Staphylococcus aureus mutation pcrA3 leads to the accumulation of pT181 replication initiation complexes.

In Staphylococcus aureus cells carrying the pcrA3 chromosomal mutation, plasmid pT181 and its derivatives were maintained at a reduced copy number. A significant proportion of their DNA migrated during agarose gel electrophoresis as nicked DNA. The results obtained in the characterization of this plasmid DNA species show that it represents replication initiation complexes. Such complexes could not be detected in a wild-type host. The replication initiation complexes present in pcrA3 cells could resume replication after a lag. It was concluded from these results that the pcrA3 host mutation affected a step in plasmid pT181 replication immediately following the formation of the replication initiation complex, and that in pcrA3 this step became rate-limiting for plasmid pT181 replication.

Bacterial Proteins↗

PubMed2026

"Tissue-specific mitochondrial dysfunction in keratoconus: An integrated structural, genomic, and functional analysis".

PURPOSE: Keratoconus (KC) is a progressive corneal ectasia characterized by stromal thinning, conical protrusion, and irregular astigmatism, leading to visual impairment. Although oxidative stress is implicated in KC, the role of mitochondrial dysfunction remains unclear. We evaluated mitochondrial structural, genomic, and functional abnormalities in corneal tissues and blood from KC patients. METHODS: This prospective study enrolled 110 KC patients and 55 controls. Transmission electron microscopy (TEM) and immunohistochemistry (IHC) were performed on epithelial and stromal tissues from 10 KC to 5 control corneas assessing mitochondrial morphology, oxidative phosphorylation (OXPHOS) complexes and pro-apoptotic protein NOXA. Whole mitochondrial DNA (mtDNA) sequencing and relative mtDNA copy number analysis were performed on paired blood and corneal tissues from 50 KC patients and 35 controls including both epithelial and stromal samples. Gene expression of mitochondrial biogenesis and oxidative stress-related genes was analysed by qRT-PCR in corneal epithelium from independent 50 KC patients and 15 controls. RESULTS: TEM revealed cristolysis, membrane disruption, and reduced mitochondrial density in KC corneas. IHC showed reduced expression of OXPHOS complexes and increased NOXA expression (p < 0.05). Sequencing identified 1107 mtDNA variants, with more variants in corneal tissues than matched blood (929 vs. 576; p = 0.0002). Recurrent likely pathogenic variants were enriched in complex I-encoding genes (ND4, ND5). KC corneas showed reduced mtDNA copy number, downregulated POLRMT, upregulated NOX4, and significant downregulation of multiple antioxidant genes (p < 0.0001). CONCLUSION: KC patients exhibit tissue-specific mitochondrial abnormalities and impaired oxidative stress regulation, supporting a role for mitochondrial dysfunction in disease pathogenesis and highlighting potential therapeutic targets.

Corneal pathology↗

PubMed1992

Characterization of a cDNA clone encoding multiple copies of the neuropeptide APGWamide in the mollusk Lymnaea stagnalis.

Male mating behavior of the simultaneous hermaphrodite freshwater snail Lymnaea stagnalis is controlled by a neuronal network that consists of various types of peptidergic neurons, as well as serotonergic cells. In the present article, we describe the isolation and characterization of a cDNA clone that encodes a multipeptide preprohormone expressed in the anterior lobe of the right cerebral ganglion, in a group of neurons that principally innervate the penial complex. The preprohormone is 219 amino acids in length and contains 10 copies of the peptide Ala-Pro-Gly-Trp-Gly. Posttranslational processing of the prohormone may lead to the generation of the amidated neuropeptide Ala-Pro-Gly-Trp-amide (APGWamide), an amidated C-terminal anterior lobe peptide, and four connecting peptide sequences, C1-C4. We show by in situ and filter hybridizations that neurons of the right anterior lobe comprise the major site of expression of the APGWamide gene. Expression of the APGWamide gene is detected in the CNS of both adult animals and noncopulating juveniles. Peptides derived from the APGWamide prohormone are probably involved in the control of a part of the male mating behavior and have both central and peripheral targets.

Amino Acid Sequence↗

PubMed1996

A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage.

We successfully isolated a rat cDNA clone encoding a novel EF hand protein with a molecular weight of about 17 kDa and designated this gene iba1 (ionized calcium binding adapter molecule 1). The genomic copy of the iba1 gene was located within a segment of the major histocompatibility complex class III region between the Bat2 and TNF alpha genes. The iba1 gene was shown to be highly expressed in testis and spleen, but weakly expressed in brain, lung, and kidney. Among brain cells, the iba1 gene was specifically expressed in microglia. A screening of hemopoietic cell lines showed that the Iba1 protein was clearly expressed in monoblastic cell lines but only very weakly expressed in myeloid cell lines. Iba1 protein is therefore suggested to act as an adapter molecule, mediating calcium signals that may function in a monocytic lineage including microglia.

Amino Acid Sequence↗

PubMed2001

Human immunodeficiency virus type 1 integrase: arrangement of protein domains in active cDNA complexes.

Early steps of retroviral replication involve reverse transcription of the viral RNA genome and integration of the resulting cDNA copy into a chromosome of the host cell. The viral-encoded integrase protein carries out the initial DNA breaking and joining reactions that mediate integration. The organization of the active integrase-DNA complex is unknown, though integrase is known to act as a multimer, and high resolution structures of the isolated integrase domains have been determined. Here we use site-specific cross-linking based on disulfide bond formation to map integrase-DNA contacts in active complexes. We establish that the DNA-binding C-terminal domain of one integrase monomer acts with the central catalytic domain from another monomer at each viral cDNA end. These data allow detailed modeling of an integrase tetramer in which pairs of trans interactions link integrase dimers bound to substrate DNA. We also detected a conformational change in integrase- DNA complexes accompanying cleavage of the viral cDNA terminus.

Amino Acid Substitution↗

PubMed1999

Identification and characterization of Schizosaccharomyces pombe asp1(+), a gene that interacts with mutations in the Arp2/3 complex and actin.

The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S. pombe arp3-c1 mutant, and have identified one, which we have termed asp1(+). The asp1(+) open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1(+) nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1(+) leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Delta strain. We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.

Actin-Related Protein 2↗

PubMed1985

A model for intracellular complexation between gene-5 protein and bacteriophage fd DNA.

A structural model for the helical intracellular complex formed between the gene-5 DNA-binding protein (G 5 BP; approximately 1274 copies) and bacteriophage fd DNA has been derived by an atomic-contact analysis approach. These studies depended in large part on the recently determined high-resolution structure of the G 5 BP dimer and cross-correlations with physical-chemical data available from other techniques. The approach was to systematically scan the full set of helical complexation parameters involved, based upon observed structural and orientational constraints, to determine those compatible with both the structure of the G 5 BP dimer and the overall dimensions of the full complex. This process was monitored throughout by close scrutiny of dimer-dimer contacts and the use of hard-copy and interactive graphics devices. Instead of the wide variety of possibilities that had been expected from such an approach, only one satisfactory assembly of DNA and G 5 BP dimers could be found. The results indicate that phage DNA will be wound to the outside of the helical protein ribbon that forms the core of intracellular complex at a density of five nucleotides per G 5 BP monomer. Bound DNA strands are positioned in two contiguous binding channels, which form as a consequence of the interactions of complexed G 5 BP dimers. These channels run just inside the outer extended beta loops, composed of residue 20-30, and are separated by approximately 3.2 nm. The DNA phosphate backbone is bound at a substantially smaller radial distance (approximately 3.5 nm) than the maximum radius of the intracellular complex as a whole (approximately 4.5 nm) since bound DNA is embedded within these well-defined binding channels. Our studies also indicate that a number of sterically unacceptable contacts, involving residues 38-42, prevent complexation of otherwise complementary dimer surfaces in the absence of nucleic acids. In the process of binding DNA, these residues change conformation thereby allowing self-assembly of dimer units into a helical structure. We propose that these residues act as a two-position stereochemical switch that allows or disallows complex formation in response to the absence or presence of DNA.

Bacteriophages↗

PubMed2004

Cytosol-derived proteins are sufficient for Arp2/3 recruitment and ARF/coatomer-dependent actin polymerization on Golgi membranes.

The actin cytoskeleton has been implicated in protein trafficking at the Golgi apparatus and in Golgi orientation and morphology. Actin dynamics at the Golgi are regulated in part by recruiting Cdc42 or Rac to the membrane through a binding interaction with the coatomer-coated (COPI)-vesicle coat protein, coatomer. This leads to actin polymerization through the effector, N-WASP and the Arp2/3 complex. Here, we have used reconstitution of vesicle budding to test whether Arp2/3 is recruited to membranes during the formation of COPI vesicles. Our results revealed that ARF1 activation leads to greatly increased Arp3 levels on the membranes. Coatomer-bound Cdc42 and pre-existing F-actin are important for Arp2/3 binding. ARF1-dependent Arp2/3 recruitment and actin polymerization can be reconstituted on liposomal membranes, indicating that no membrane proteins are necessary. These results show that activated ARF1 can stimulate Arp2/3 recruitment to Golgi membranes through coatomer, Cdc42 or Rac, and N-WASP.

ADP-Ribosylation Factor 1↗

PubMed1992

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II).

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.

Bacillus subtilis↗

PubMed2001

The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry.

We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E. coli. It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs. At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1. The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques. This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.

Carbohydrate Sequence↗

PubMed1997

Molecular characterization of a gene in the rat homologous to human CD94.

Three classes of major histocompatibility (MHC) class I binding receptors on natural killer (NK) cells have so far been described: CD94/NKG2 heterodimeric receptors and killer cell inhibitory receptors in the human, and Ly-49 homodimers in rodents. CD94, NKG2 and Ly-49 belong to the C-type lectin superfamily. As yet, CD94 and NKG2 molecules have not been detected in rodents or Ly-49 in humans. It has therefore been proposed that the two receptors represent functional equivalents in these species. The present study describes the cDNA cloning of a novel rat gene encoding a protein of 179 amino acids, 54.2% identical to human CD94. The single-copy Cd94 gene is localized to the rat NK gene complex (NKC), within 50 kb from Nkrp2, between the Nkrp1 and Ly49 gene clusters. By Northern blot analysis, we showed that rat CD94 is selectively expressed by NK cells and a small subset of T cells, similar to the human orthologue. This expression is strain dependent, with high expression in DA NK cells and low in PVG NK cells. Evidence is presented that this difference is not due to receptor repertoire shaping by MHC-encoded ligands, but is controlled by genetic elements residing within the NKC. The identification of a rat CD94 orthologue suggests that NK cell populations utilize two different C-type lectin receptors for MHC class I molecules in parallel.

Amino Acid Sequence↗

PubMed2000

Mitochondrial genome-encoded ATPase subunit 6+8 mRNA increases in human hepatoblastoma cells in response to nonfatal cold stress.

Cellular responses to cold stress have not been well clarified, compared with heat shock responses, especially in mammalian cells. We investigated cold-stress responses in human hepatoblastoma cells (HepG2) exposed to a nonfatal temperature of 17 degrees C. Under the condition, RNA and protein syntheses in the cells were highly, but incompletely, depressed and cell growth was impaired. A cDNA subtraction method was used to isolate mRNAs for which the levels were increased in cold-stressed cells compared with cells cultured at 37 degrees C. A transcript isolated by the screening was identified as ATPase subunit 6+8 mRNA that encodes components of a mitochondrial ATPase complex and that is transcribed from a mitochondrial genome. The copy number of the mitochondrial genome in cells was not changed by cold stress. Thus, HepG2 cells were treated with various concentrations of actinomycin D and chloramphenicol to assess the effects of transcriptional and translational reduction on the increased level of the ATPase subunit 6+8 mRNA. The mRNA level was increased in cells treated with low concentrations of the RNA or protein synthesis inhibitors. These results indicate that the increase in ATPase subunit 6+8 mRNA stimulated by cold stress could be mediated by a partial decline of transcription and/or translation in the cells. In addition, the degradation of ATPase subunit 6+8 mRNA was suppressed in cold-stressed cells compared with that in 37 degrees C-cultured cells. This result implies that posttranscriptional regulation is also involved in the cold-stimulated increase in ATPase subunit 6+8 mRNA in HepG2 cells.

Apoptosis↗