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Genetic recombination and the cell cycle: what we have learned from chromosome dimers.

Genetic recombination is central to DNA metabolism. It promotes sequence diversity and maintains genome integrity in all organisms. However, it can have perverse effects and profoundly influence the cell cycle. In bacteria harbouring circular chromosomes, recombination frequently has an unwanted outcome, the formation of chromosome dimers. Dimers form by homologous recombination between sister chromosomes and are eventually resolved by the action of two site-specific recombinases, XerC and XerD, at their target site, dif, located in the replication terminus of the chromosome. Studies of the Xer system and of the modalities of dimer formation and resolution have yielded important knowledge on how both homologous and site-specific recombination are controlled and integrated in the cell cycle. Here, we briefly review these advances and highlight the important questions they raise.

Bacteria↗

The eukaryotic translation initiation factor 4E confers multiallelic recessive Bymovirus resistance in Hordeum vulgare (L.).

Virus diseases are widespread threats for crop production, which can, in many cases, be controlled efficiently by exploiting naturally occurring resistance. Barley, an important cereal species of the Triticeae, carries two genes, rym4 and rym5, which are located in the telomeric region of chromosome 3HL and confer recessive resistance to various strains of the Barley yellow mosaic virus complex. The barley 'eukaryotic translation initiation factor 4E' (Hv-eIF4E) was identified as a candidate for resistance gene function by physical mapping on a 650 kb contig. It is located in a chromosomal region characterized by suppressed recombination, in a position collinear to its homologue on rice chromosome 1L. Sequence diversity in the coding region of Hv-eIF4E, as calculated from a collection of unrelated barley accessions, revealed non-silent single nucleotide polymorphisms (SNPs) in four of its five exons. Stable transformation of a resistant barley genotype with a genomic fragment or a full-length cDNA of Hv-eIF4E derived from susceptible cultivars induced susceptibility to Barley mild mosaic virus. Moreover, the identification of SNPs diagnostic for rym4 and rym5 provides evidence that these are two alleles, which confer different resistance specificities. These findings demonstrate that variants of Hv-eIF4E confer multiallelic recessive virus resistance in a monocot species. The identification of eIF4E as the causal host factor for bymovirus resistance illustrates that mutations in this basic component of the eukaryotic translation complex form a seminal mechanism for recessive virus resistance in both dicot and monocot plants.

Alleles↗

A simple and rapid micromethod for genomic DNA extraction from jugal epithelial cells. Application to human lymphocyte antigen typing in one large family of atopic/asthmatic probands.

We describe a rapid and reliable micromethod for DNA isolation from buccal epithelial cells from the interior mouth mucosa. This convenient, noninvasive method could be applied to genetic typing in a small number of cells (about 2000 cells per cheek). We have shown that DNA released by this method is suitable for further amplification by polymerase chain reaction (PCR). Using this protocol, coupled with the PCR-RFLP (restriction fragment length polymorphism) method, we analyzed the allelic sequence diversity of the human lymphocyte antigen (HLA) class II genes in an extended family of 33 persons containing 14 asthmatic or atopic members. Six of eight DQA1 alleles, and 11 DQB1, 20 DPB1, and 10 DR haplotypes could be identified in a single DNA sample. Our results suggest that the DR53 group haplotype is frequently associated with allergic asthma and atopy. The micromethod described here may be useful in genetic epidemiology, especially in family studies involving small children.

Asthma↗

Molecular variation of the human elastin (ELN) gene in a normal human population.

DNA sequence diversity in the human elastin genomic region has been estimated by RFLP analysis in a normal human population. The proportion of polymorphic nucleotides and the degree of nucleotide diversity were 0.0034 and 0.0018 respectively. It is argued that the estimate of nucleotide diversity does not indicate strong purifying selection in the region. A total of 144 restriction sites were sampled in each of 80 independent chromosomes representing the screening of 58080 bp overall. Six main haplotypes were constructed; they represent at least 84% of the 80 chromosomes sampled. Analysis for linkage disequilibrium revealed two statistically significant comparisons out of 54 tests, approximately the proportion that would be statistically significant at the 5% level by chance. A higher order quadrigenic disequilibrium was detected. The relationship between the physical distance separating polymorphic restriction sites and linkage disequilibrium is discussed. The development of elastin haplotypes and knowledge of the pattern of linkage disequilibrium should aid the study of elastin related disease and human evolution.

Blotting, Southern↗

The biological degradation of cellulose.

Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the beta-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.

Biodegradation, Environmental↗

Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation.

The oxyR gene positively regulates genes induced by oxidative stress in Salmonella typhimurium and Escherichia coli. Purification of the OxyR protein showed that oxidized but not reduced OxyR activates transcription of oxidative stress-inducible genes in vitro. Conversion between the two forms of OxyR is rapid and reversible. Both the oxidized and the reduced forms of the OxyR protein are capable of binding to three diverse sequences upstream of OxyR-regulated promoters, but the interactions of the two forms of OxyR with the promoter regions are different. The results suggest that direct oxidation of the OxyR protein brings about a conformational change by which OxyR transduces an oxidative stress signal to RNA polymerase.

Bacterial Proteins↗

Modulation of folding pathways of exported proteins by the leader sequence.

Leader peptides that function to direct export of proteins through membranes have some common features but exhibit a remarkable sequence diversity. Thus there is some question whether leader peptides exert their function through conventional stereospecific protein-protein interaction. Here it is shown that the leader peptides retarded the folding of precursor maltose-binding protein and ribose-binding protein from Escherichia coli. This kinetic effect may be crucial in allowing precursors to enter the export pathway.

ATP-Binding Cassette Transporters↗

bla(VIM-2) cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital.

We investigated the phenotypic and genetic properties of metallo-beta-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 beta-lactamase, a class B enzyme previously found only in P. aeruginosa isolates from Europe. VIM-2-producing isolates of Pseudomonas putida were also detected. Resistance was transferable from both these species to P. aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing P. aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P. putida isolates were diverse. Sequencing showed that the bla(VIM-2) genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a P. aeruginosa isolate had bla(VIM) located downstream of a variant of aacA4. bla(VIM) also lay in a class 1 integron in a representative P. putida strain, but the organization of this integron was different from that sequenced from the P. aeruginosa strain. In conclusion, the metallo-beta-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance.

Conjugation, Genetic↗

Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization.

In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

Animals↗

Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons.

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae.Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10(4) CFU g of soil(-1). The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.

Base Sequence↗

Passive transfer of antiserum specific for immunogens derived from a nontypeable Haemophilus influenzae adhesin and lipoprotein D prevents otitis media after heterologous challenge.

We recently determined that passive transfer of serum directed against a synthetic peptide called LB1 or a recombinant fusion protein immunogen [LPD-LB1(f)(2,1,3)] could prevent otitis media after challenge with a homologous nontypeable Haemophilus influenzae (NTHI) isolate. NTHI residing in the nasopharynx was rapidly cleared from this site, thus preventing it from ascending the eustachian tube and inducing otitis media in chinchillas compromised by an ongoing viral upper respiratory tract infection. While LB1 is based solely on one NTHI adhesin, the latter immunogen, LPD-LB1(f)(2,1,3), was designed to incorporate two NTHI antigens shown to play a role in the pathogenesis of otitis media; lipoprotein D (LPD) and the P5-homologous fimbrin adhesin. The design of LPD-LB1(f)(2,1,3) also accommodated for the recently demonstrated existence of three major groupings, based on amino acid sequence diversity, in the third surface-exposed region of P5-fimbrin. LPD-LB1(f)(2,1,3) was thus designed to potentially confer broader protection against challenge by diverse strains of NTHI. Chinchillas were passively immunized here with serum specific for either LB1 or for LPD-LB1(f)(2,1,3) prior to challenge with a member of all three groups of NTHI relative to diversity in region 3. The transferred serum pools were also analyzed for titer, specificity, and several functional activities. We found that both serum pools had equivalent ability to mediate C'-dependent killing and to inhibit adherence of NTHI strains to human oropharyngeal cells. When passively transferred, both serum pools significantly inhibited the signs and incidence of otitis media (P </= 0.01) induced by any of the three challenge isolates. Despite providing protection against disease, the ability of these antisera to induce total eradication of NTHI from the nasopharynx was not equivalent among NTHI groups. These data thus suggested that while early, complete eradication of NTHI from the nasopharynx was highly protective, reduction of the bacterial load to below a critical threshold level appeared to be similarly effective.

Adhesins, Bacterial↗

Specificity of the protective antibody response to apical membrane antigen 1.

Apical membrane antigen 1 (AMA1) is considered one of the leading candidates for inclusion in a vaccine against blood stages of Plasmodium falciparum. Although the ama1 gene is relatively conserved compared to those for some other potential vaccine components, numerous point mutations have resulted in amino acid substitutions at many sites in the polypeptide. The polymorphisms in AMA1 have been attributed to the diversifying selection pressure of the protective immune responses. It was therefore of interest to investigate the impact of sequence diversity in P. falciparum AMA1 on the ability of anti-AMA1 antibodies to inhibit the invasion of erythrocytes in vitro by P. falciparum merozoites. For these studies, we used antibodies to recombinant P. falciparum 3D7 AMA1 ectodomain, which was prepared for testing in early clinical trials. Antibodies were raised in rabbits to the antigen formulated in Montanide ISA720, and human antibodies to AMA1 were isolated by affinity purification from the plasma of adults living in regions of Papua New Guinea where malaria is endemic. Both rabbit and human anti-AMA1 antibodies were found to be strongly inhibitory to the invasion of erythrocytes by merozoites from both the homologous and two heterologous lines of P. falciparum. The inhibitory antibodies targeted both conserved and strain-specific epitopes within the ectodomain of AMA1; however, it appears that the majority of these antibodies reacted with strain-specific epitopes in domain I, the N-terminal disulfide-bonded domain, which is the most polymorphic region of AMA1.

Animals↗

Identification of a genetically distinct subspecies of Mycobacterium kansasii.

To assess the usefulness of a specific DNA probe for Mycobacterium kansasii, 105 isolates from Australia, Belgium, Japan, South Africa, and Switzerland were collected and analyzed. Twenty of these isolates were probe negative, of which 18 were from Belgium and Switzerland. Analysis of all isolates by Southern blot hybridization indicated a lack of variability among probe-positive isolates, while probe-negative isolates were clearly distinct and showed greater diversity. Sequence analysis of the 250 nucleotides at the 5' end of the 16S rRNA gene revealed that 19 of the 20 probe-negative isolates had a sequence different from that of M. kansasii. A total of five nucleotide differences were present in a cluster consisting of two nucleotide deletions and three nucleotide substitutions. These results suggest the existence of a genetic subspecies of M. kansasii.

Base Sequence↗

Structural insights into adeno-associated virus serotype 5.

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-&#xc5; resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between &#x3b2;H and &#x3b2;I of the core &#x3b2;-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.

Capsid Proteins↗

Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys.

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4(+) T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.

AIDS Vaccines↗

Characterization of human chromosomal constitutive heterochromatin.

The constitutive heterochromatin of human chromosomes is evaluated by various selective staining techniques, i.e., CBG, G-11, distamycin A plus 4,6-diamidino-2-phenylindole-2-HCl (DA/DAPI), the fluorochrome D287/170, and Giemsa staining following the treatments with restriction endonucleases AluI and HaeIII. It is suggested that the constitutive heterochromatin could be arbitrarily divided into at least seven types depending on the staining profiles expressed by different regions of C-bands. The pericentromeric C-bands of chromosomes 1, 5, 7, 9, 13-18, and 20-22 consist of more than one type of chromatin, of which chromosome 1 presents the highest degree of heterogeneity. Chromosomes 3 and 4 show relatively less consistent heterogeneous fractions in their C-bands. The C-bands of chromosomes 10, 19, and the Y do not have much heterogeneity but have characteristic patterns with other methods using restriction endonucleases. Chromosomes 2, 6, 8, 11, 12, and X have homogeneous bands stained by the CBG technique only. Among the chromosomes with smaller pericentric C-bands, chromosome 18 shows frequent heteromorphic variants for the size and position (inversions) of the AluI resistant fraction of C-band. The analysis of various types of heterochromatin with respect to specific satellite and nonsatellite DNA sequences suggest that the staining profiles are probably related to sequence diversity.

Amniotic Fluid↗

Evolution of antigen binding receptors.

This review addresses issues related to the evolution of the complex multigene families of antigen binding receptors that function in adaptive immunity. Advances in molecular genetic technology now permit the study of immunoglobulin (Ig) and T cell receptor (TCR) genes in many species that are not commonly studied yet represent critical branch points in vertebrate phylogeny. Both Ig and TCR genes have been defined in most of the major lineages of jawed vertebrates, including the cartilaginous fishes, which represent the most phylogenetically divergent jawed vertebrate group relative to the mammals. Ig genes in cartilaginous fish are encoded by multiple individual loci that each contain rearranging segmental elements and constant regions. In some loci, segmental elements are joined in the germline, i.e. they do not undergo genetic rearrangement. Other major differences in Ig gene organization and the mechanisms of somatic diversification have occurred throughout vertebrate evolution. However, relating these changes to adaptive immune function in lower vertebrates is challenging. TCR genes exhibit greater sequence diversity in individual segmental elements than is found in Ig genes but have undergone fewer changes in gene organization, isotype diversity, and mechanisms of diversification. As of yet, homologous forms of antigen binding receptors have not been identified in jawless vertebrates; however, acquisition of large amounts of structural data for the antigen binding receptors that are found in a variety of jawed vertebrates has defined shared characteristics that provide unique insight into the distant origins of the rearranging gene systems and their relationships to both adaptive and innate recognition processes.

Amphibians↗

Evolution of Wheat streak mosaic virus: dynamics of population growth within plants may explain limited variation.

Like many other plant RNA viruses, Wheat streak mosaic virus (WSMV) sequence diversity within and among infected plants is low given the large number of virions produced. This may be explained by considering aspects of plant virus life history. Intracellular replication of RNA viruses is predominately linear, not exponential, which means that the rate at which mutations accumulate also is linear. Bottlenecks during systemic movement further limit diversity. Analysis of mixed infections with two WSMV isolates suggests that about four viral genomes participate in systemic invasion of each tiller. Low effective population size increases the role of stochastic processes on dynamics of plant virus population genetics and evolution. Despite low pair-wise diversity among isolates, the number of polymorphic sites within the U.S. population is about the same as between divergent strains or a sister species. Characteristics of polymorphism in the WSMV coat protein gene suggest that most variation appears neutral.

Biological Evolution↗