Large-scale identification of polymorphic microsatellites using an <Emphasis Type="Italic">in silico</Emphasis> approach

Background  

Simple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.     

Primary structure characterization of a Rhodocyclus tenuis diheme cytochrome c reveals the existence of two different classes of low-potential diheme cytochromes c in purple phototropic bacteria

The complete amino acid sequence of a 26-kDa low redox potential cytochrome c-551 from Rhodocyclus tenuis was determined by a combination of Edman degradation and mass spectrometry. There are 240 residues including two heme binding sites at positions 41, 44, 128, and 132. There is no evidence for gene doubling. The only known homolog of Rc. tenuis cytochrome c-551 is the diheme cytochrome c-552 from Pseudomonas stutzeri which contains 268 residues and heme binding sites at nearly identical positions. There is 44% overall identity between the Rc. tenuis and Ps. stutzeri cytochromes with 10 internal insertions and deletions. The Ps. stutzeri cytochrome is part of a denitrification gene cluster, whereas Rc. tenuis is incapable of denitrification, suggesting different functional roles for the cytochromes. Histidines at positions 45 and 133 are the fifth heme ligands and conserved histidines at positions 29, 209, and 218 and conserved methionines at positions 114 and 139 are potential sixth heme ligands. There is no obvious homology to the low-potential diheme cytochromes characterized from other purple bacterial species such as Rhodobacter sphaeroides. There are therefore at least two classes of low-potential diheme cytochromes c found in phototrophic bacteria. There is no more than 11% helical secondary structure in Rc. tenuis cytochrome c-551 suggesting that there is no relationship to class I or class II c-type cytochromes.     

Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.     

A high risk phenotype of hypertrophic cardiomyopathy associated with a compound genotype of two mutated β-myosin heavy chain genes

Hypertrophic cardiomyopathy (HCM) is a genetically and clinically heterogeneous myocardial disease that is in most cases familial and transmitted in a dominant fashion. The most frequently affected gene codes for the cardiac (ventricular) β-myosin heavy chain. We have investigated the genetic cause of an isolated case of HCM, which was marked by an extremely severe phenotype and a very early age of onset. HCM is normally not a disease of small children. The proband was a boy who had suffered cardiac arrest at the age of 6.5years (resuscitation by cardioconversion). Upon screening of the β-myosin heavy chain gene as a candidate, two missense mutations, one in exon19 (Arg719Trp) and a second in exon12 (Met349Thr), were identified. The Arg719Trp mutation was de novo, as it was not found in the parents. In contrast, the Met349Thr mutation was inherited through the maternal grandmother. Six family members were carriers of this mutation but only the proband was clinically affected. Segregation and molecular analysis allowed us to assign the Met349Thr mutation to the maternal and the Arg719Trp de novo mutation to the paternal β-myosin allele. Thus, the patient has no normal myosin. We interpret these findings in terms of compound heterozygosity of a dominant (Arg719Trp) and a recessive (Met349Thr) mutation. Whereas a single mutated Arg719Trp allele would be sufficient to cause HCM, the concurrent Met349Thr mutation alone does not apparently induce the disease. Nevertheless, it conceivably contributes to the particularly severe phenotype. Received: 15 September 1997 / Accepted: 26 November 1997     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

The frequency of multiple recombination events occurring at the human Ig kappa L chain locus

Products of Ig kappa L chain gene rearrangement in a variety of human B cell samples were investigated by sequential Southern blot hybridization analysis. By application of four region-specific probes (C kappa, J kappa, U' kappa and kappa de) a complete spectrum of kappa rearrangements, including both predicted and novel products, were detected. Nearly 30% of the products detected reflect multiple recombination of the kappa locus. The kappa-deleting element was responsible for 70% of the multiple rearrangements that were detected. Interestingly, eight kappa-expressing samples exhibited rearrangement of the kappa-deleting element. The remaining multiple recombination products were characteristic of double V kappa-J kappa rearrangement. This frequency reveals that secondary V-J rearrangement may significantly contribute to the expression of kappa L chains in humans.     

Assignment of the Charcot-Marie-Tooth neuropathy type 1 (CMT 1a) gene to 17p11.2-p12.

Charcot-Marie-Tooth disease type 1a (CMT 1a) is an autosomal dominant peripheral neuropathy linked to the DNA markers D17S58 and D17S71, located in the pericentromeric region of the chromosome 17p arm. We analyzed an extended 5-generation Belgian family, multiply affected with CMT 1a, for linkage with eight chromosome 17 markers. The results indicated that the CMT 1a mutation is localized in the chromosomal region 17p11.2-p12 between the marker D17S71 and the gene for myosin heavy polypeptide 2 of adult skeletal muscle.     

Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.The most common type of adhesive organelle in the Enterobacteriaceae is the type 1 fimbria, which has been most extensively studied in Escherichia coli. The corresponding structures of Klebsiella pneumoniae are similar to those of E. coli with regard to genetic composition and regulation (15). Type 1 fimbriae are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific adhesin FimH. The FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress (48).It has been demonstrated in E. coli that FimH has two domains, the mannose-binding lectin domain (from amino acid [aa] 1 through 156) and the fimbria-incorporating pilin domain (from aa 160 through 279), connected via a 3-aa-long linker chain (6). A mannose-binding site is located at the top of the lectin domain, at the opposite end from the interdomain linker (17).Several studies have demonstrated that type 1 fimbriae play an important role in E. coli urinary tract infection (UTI) (7, 21, 23, 35). In addition, in urinary E. coli isolates, the FimH adhesin accumulates amino acid replacements which increase tropism for the uroepithelium and various components of basement membranes (21, 30, 35, 37, 49). Most of the replacements increase the monomannose binding capability of FimH under low shear, by altering allosteric catch bond properties of the protein (48). The mutated FimH variants were shown to provide an advantage in colonization of the urinary tract in the mouse model (35) and correlate with the overall extraintestinal virulence of E. coli (16). Thus, FimH mutations are pathoadaptive in nature.Klebsiella pneumoniae is recognized as an important opportunistic pathogen frequently causing UTIs, septicemia, or pneumonia in immunocompromised individuals (29). It is responsible for up to 10% of all nosocomial bacterial infections (18, 41). K. pneumoniae is ubiquitous in nature, and it has been shown that environmental isolates are phenotypically indistinguishable from clinical isolates (22, 26, 27, 29, 33). Furthermore, it has been demonstrated that environmental isolates of K. pneumoniae are as virulent as clinical isolates (28, 45).K. pneumoniae possesses a number of known virulence factors, including a pronounced capsule, type 3 fimbriae, and type 1 fimbriae (29, 44). Type 1 fimbriae produced by K. pneumoniae are described as functionally and structurally similar to type 1 fimbriae from E. coli (25) and have been shown to play a significant role in K. pneumoniae UTI (32, 43).We have previously shown that mature FimH from 54 isolates of K. pneumoniae (isolated from urine, blood, liver, and the environment) is represented by seven protein variants due to point amino acid replacements. (42) When K. pneumoniae FimH was aligned with the FimH of E. coli, they showed ∼85% similarity at the amino acid level. Furthermore, a majority (14 out of 21 isolates) of the K. pneumoniae strains isolated from patients with UTI grouped into a single clonal group based on multilocus sequence typing, but fimH in one isolate in the group differed from the others by a single nucleotide mutation resulting in an amino acid change, serine to alanine, in position 62 (42). The same mutation has been found in FimH of a highly uropathogenic clone of E. coli and significantly increases the adhesin''s ability to adhere to monomannose under low or no shear (19, 39, 50).In this study, we describe the extent and pattern of structural variability of the FimH protein from K. pneumoniae and perform comparative analyses of the functional properties of FimH from both K. pneumonae and E. coli.     

Phototropin‐ and photosynthesis‐dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue‐light‐dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue‐light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop‐and‐go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma‐membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2‐ and photosynthesis‐dependent signals. The present study might add novel regulatory mechanism for light‐dependent plant organelle interactions.