Structural rearrangements of the central region of the morbillivirus attachment protein stalk domain trigger F protein refolding for membrane fusion

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

Control of chromosomal rearrangements in {\it Escherichia coli\/}

A complete physical map of Escherichia coli K-12 strain MG1655 was constructed by digesting chromosomal DNA with the infrequently cutting restriction enzymes NotI, SfiI and XbaI and separating the fragments by pulsed field gel electrophoresis. The map was used to compare six K-12 strains of E. coli. Although several differences were noted and localized, the map of MG1655 was representative of all the K-12 strains tested. The maps were also used to analyze chromosomal rearrangements in the E. coli strain MG1655. The spontaneous and UV induced frequencies of tandem duplication formation were measured at several loci distributed around the chromosome. The spontaneous duplication frequency varied from 10$\sp{-5}$ to 10$\sp{-3}$ and increased at least ten-fold following mild UV irradiation treatment. Duplications of several regions of the chromosome, including the serA region and the metE region, were mapped using pulsed field gel electrophoresis. Duplications of serA were found to be large, ranging in size from 600 kb to 2100 kb. Several of the duplications isolated at serA were caused by ectopic recombination between IS5 elements and between IS186 elements. Duplications of the metE region, however, were almost exclusively the result of ectopic recombination between ribosomal RNA cistrons. Duplication frequencies were determined at both serA and metE in wild type and mismatch repair mutant strains (mutL, mutS, uvrD and recF). Even though all of the mismatch repair mutations increased duplication frequency of metE, the largest increases were observed in the mutL and mutS strains. Duplication frequency of serA was increased less dramatically by mutations in mismatch repair. Several duplications of metE isolated in a wild type and a mismatch repair mutant were mapped. The results showed that the same repeated sequences were used for duplication formation in the mismatch repair mutant as were used in the wild type strain. Several isolates showed evidence of multiple rearrangements indicating that mismatch repair may play a role in stabilizing the genome by controlling chromosomal rearrangement. ^

Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44+CD25+ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44−CD25+ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4+CD8+ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Deregulated expression of c-Myc in a translocation-negative plasmacytoma on extrachromosomal elements that carry IgH and myc genes

The induced expression of c-Myc in plasmacytomas in BALB/c mice is regularly associated with nonrandom chromosomal translocations that juxtapose the c-myc gene to one of the Ig loci on chromosome 12 (IgH), 6 (IgK), or 16 (IgL). The DCPC21 plasmacytoma belongs to a small group of plasmacytomas that are unusual in that they appear to be translocation-negative. In this paper, we show the absence of any c-myc-activating chromosomal translocation for the DCPC21 by using fluorescent in situ hybridization, chromosome painting, and spectral karyotyping. We find that DCPC21 harbors c-myc and IgH genes on extrachromosomal elements (EEs) from which c-myc is transcribed, as shown by c-myc mRNA tracks and extrachromosomal gene transfer experiments. The transcriptional activity of these EEs is supported further by the presence of the transcription-associated phosphorylation of histone H3 (H3P) on the EEs. Thus, our data suggest that in this plasmacytoma, c-Myc expression is achieved by an alternative mechanism. The expression of the c-Myc oncoprotein is initiated outside the chromosomal locations of the c-myc gene, i.e., from EEs, which can be considered functional genetic units. Our data also imply that other “translocation-negative” experimental and human tumors with fusion transcripts or oncogenic activation may indeed carry translocation(s), however, in an extrachromosomal form.

Structural assignments to the Mycoplasma genitalium proteins show extensive gene duplications and domain rearrangements

The parasitic bacterium Mycoplasma genitalium has a small, reduced genome with close to a basic set of genes. As a first step toward determining the families of protein domains that form the products of these genes, we have used the multiple sequence programs psi-blast and geanfammer to match the sequences of the 467 gene products of M. genitalium to the sequences of the domains that form proteins of known structure [Protein Data Bank (PDB) sequences]. PDB sequences (274) match all of 106 M. genitalium sequences and some parts of another 85; thus, 41% of its total sequences are matched in all or part. The evolutionary relationships of the PDB domains that match M. genitalium are described in the structural classification of proteins (SCOP) database. Using this information, we show that the domains in the matched M. genitalium sequences come from 114 superfamilies and that 58% of them have arisen by gene duplication. This level of duplication is more than twice that found by using pairwise sequence comparisons. The PDB domain matches also describe the domain structure of the matched sequences: just over a quarter contain one domain and the rest have combinations of two or more domains.

A model for spontaneous B-lineage lymphomas in IgHμ-HOX11 transgenic mice

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR α/δ regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHμ-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin’s lymphoma of peripheral mature B cell origin.

A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

CMS: An adapter molecule involved in cytoskeletal rearrangements

Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130Cas. CMS contains three SH3 in its NH2 terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130Cas, Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130Cas to membrane ruffles and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.

Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G0 fibroblasts were either preheated (45°C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 × 10–3 dsb/Gy/Mb. Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12–16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular.

Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma.

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.

Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins.

The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.

Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139.

The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.

Facile ketene-ketene and ketene-ketenimine rearrangements: A study of the 1,3-migration of alpha-substituents interconverting alpha-imidoylketenes and alpha-oxoketenimines, a pseudopericyclic reaction

Theoretical calculations (B3LYP/6-311+G(3df,2p)//B3LYP/6-31G*) of the 1,3 migration of NR2 transforming alpha-oxoketenimines 1 to alpha-imidoylketenes 3 and vice versa indicate that this process is a pseudo-pericyclic reaction with a low activation energy (NH2 97 kJ mol(-1), N(CH3)(2) 62 kJ mol(-1)). The oxoketenimines were found to be more stable (by 18-35 kJ mol(-1)) which is in line with experimental observations. The hindered amine rotation in the amide and amidine moieties adjacent to the cumulenes are important in the migration of the NR2 group, as one of the rotation transition states is close to the 1,3 migration pathway. This gives an interesting potential energy surface with a valley-ridge inflection (VRI) between the orthogonal hindered amine rotation and 1,3 migration transition states. The imidoylketene may also undergo ring closure to an azetinone 5; however, this is metastable, and under the conditions that allow the 1,3-migration, the oxoketenimine 1 will be favored. The imine NH E/Z-interconversion of the ketenimine group takes place by inversion and has a low activation barrier (similar to40 kJ mol(-1)). In all the amidines examined the E/Z-interconversion of the imine function was predicted to be by rotation with a high barrier (>80 kJ mol(-1)), in contrast to all other reported imine E/Z-interconversions which are by inversion.