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[Multilocus VNTR-typing of Francisella tularensis strains].

In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis.

Alleles↗

Maintaining genetic stability through TP53 mediated checkpoint control.

TP53 serves as a key relay for signals elicited by cellular stresses arising from diverse environmental or therapeutic insults. This relay then activates a cell cycle arrest or cell death program, depending on the stimulus and cell type. The absence of TP53 function disables the cell death or arrest programmes, thereby allowing the emergence of variants with various types of genomic alterations. The data discussed focus on two different types of signals that trigger the TP53 relay system. Firstly, TP53 arrests cell cycle progression in response to the types of DNA damage most commonly detected in cells undergoing tumour progression. Secondly, TP53 is activated by specific depletion of ribonucleotide pools, which prevent cells from entering S phase under conditions that could lead to chromosome breakage. The contribution of both responses limits the emergence of genetic variants. The DNA damage induced arrest appears to be triggered by as few as one double strand break in normal human fibroblasts. Analysis of the arrest kinetics after ionizing radiation shows that TP53 activates a prolonged arrest response in cells with irreparable DNA damage and that high efficiency cell elimination is achieved by a process that can be activated over multiple cell cycles. These data indicate that the primary function of the TP53 arrest/apoptosis pathway in response to double strand break is to eliminate damaged cells from the proliferating population, not to allow additional time for lesion repair. However, it remains possible that repair of other types of damage may benefit from TP53 mediated arrest. Analyses in model genetic systems indicate that the absence of TP53 function allows, but does not ensure, a high intrinsic rate of genetic variation and that instability is increased substantially when cells proceed through S phase under inappropriate growth conditions. This implies that inactivation of TP53 function in combination with other genetic alterations, such as oncogene activation, could accelerate genomic instability and tumour progression.

DNA Damage↗

Test of linkage and/or association between the estrogen receptor alpha gene with bone mineral density in Caucasian nuclear families.

Extensive studies have been performed on the association between the estrogen receptor alpha (ER-alpha) gene and bone mineral density (BMD). Despite considerable efforts, the studies using limited markers and relatively small sample size have yielded largely inconsistent results. In this study, 1873 Caucasian subjects from 405 nuclear families containing 1512 sib pairs were recruited. BMD at the lumbar spine (LS) and femoral neck (FN) was measured by dual-energy X-ray absorptiometry (DXA). Seven single-nucleotide polymorphisms (SNPs) spanning from exon 1 to 8 in the ER-alpha gene were genotyped. The program QTDT (quantitative transmission disequilibrium test) was applied to test linkage and/or association of the ER-alpha gene and BMD variation using individual SNP markers and reconstructed haplotypes. Linkage disequilibrium (LD) was generally detected for SNPs in the ER-a gene (P < 0.05). Associations were observed between SNP rs932477 and FN BMD (P = 0.028), and between the most predominant three-marker haplotype (GCG) containing SNP rs932477 and FN BMD (P = 0.010). Within-family association (present only with both linkage and association) between SNP rs2228480 (G2014A) and FN BMD (P = 0.015) was observed. The most predominant seven-SNP haplotype (TCGCGGG) was associated with higher LS BMD (P = 0.015). However, after correction for multiple testing, these associations did not reach statistical significance. Denser markers may be necessary to better define the relationship between the ER-alpha gene and BMD variation in our sample.

Absorptiometry, Photon↗

A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries.

A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 10(9) sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an alpha-synuclein ep-PCR library and NNS synthetic oligonucleotide libraries are given as examples.

Algorithms↗

A resource to acquire and summarize pharmacogenetics knowledge in the literature.

To determine how genetic variations contribute the variations in drug response, we need to know the genes that are related to drugs of interest. But there are no publicly available data-bases of known gene-drug relationships, and it is time-consuming to search the literature for this information. We have developed a resource to support the storage, summarization, and dissemination of key gene-drug interactions of relevance to pharmacogenetics. Extracting all gene-drug relationships from the literature is a daunting task, so we distributed a tool to acquire this knowledge from the scientific community. We also developed a categorization scheme to classify gene-drug relationships according to the type of pharmacogenetic evidence that supports them. Our resource (http://www.pharmgkb.org/home/project-community.jsp) can be queried by gene or drug, and it summarizes gene-drug relationships, categories of evidence, and supporting literature. This resource is growing, containing entries for 138 genes and 215 drugs of pharmacogenetics significance, and is a core component of PharmGKB, a pharmacogenetics knowledge base (http://www.pharmgkb.org).

Databases, Bibliographic↗

The role of recombination and mutation in 16S-23S rDNA spacer rearrangements.

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.1 4.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.

Bacteria↗

Diversity, distribution and divergence of lin genes in hexachlorocyclohexane-degrading sphingomonads.

Two forms of hexachlorocyclohexane (HCH), gamma-HCH (lindane) and technical HCH (incorporating alpha-, beta-, gamma- and delta- isomers), have been used against agricultural pests and in health programs since the 1940s. Although all the isomers are present in the milieu, delta- and beta-HCH isomers are the most problematic and present a serious environmental problem. Bacteria that degrade HCH isomers have been isolated from HCH contaminated soils from different geographical locations around the world (from the family Sphingomonadaceae). Interestingly, all these bacteria contain nearly identical lin genes (responsible for HCH degradation), which are diverging to perform several catabolic functions. The organization and diversity of lin genes have been studied among several sphingomonads, and they have been found to be associated with plasmids and IS6100, both of which appear to have a significant role in their horizontal transfer. The knowledge of the molecular genetics, diversity and distribution of lin genes, and the potential of sphingomonads to degrade HCH isomers, can now be used for developing bioremediation techniques for the decontamination of HCH contaminated sites.

Bacterial Proteins↗

Pubertal traits and seasonal variation of the sexual activity in Brahman, Hereford and crossbred heifers.

Age and weight at puberty and the pattern of ovulatory activity from puberty to 26 mo of age were studied in a total of 55 Brahman (B), Hereford (H), 5/8B-3/8H (B(5)H(3)) and 5/16B-11/16H (B(5)H(11)) heifers. The trial was conducted over two years. Heifers were kept during the whole experimental period on a paddock of native pasture at a stocking rate of 0.5 animals per hectare. Vasectomized bulls were used for estrus detection and blood was collected from the heifers twice weekly to monitor ovulatory activity through progesterone level as determinated by RIA. In addition to age and weight at puberty, the weights and dates at the time of cessation and resumption of ovulatory activity were also considered. There were significant effects (P<0.05 or P<0.01) of genetic group (purebreds vs crossbreds) for age and weight at puberty; the difference being 60 d and 18 kg, respectively, favoring the crossbred groups. After puberty attainment, an anestrous period was detected in all Brahman (118.6 d) and B(5)H(3) (113.4 d) heifers; anestrus was observed in only 14% of B(5)H(11) heifers and for a shorter (43.5 days) period. Hereford heifers did not exhibit interruption of ovulatory activity. In Brahman and B(5)H(3) heifers, the pattern of interruption and resumption of estrus and ovulatory activity correlated closely (0.89 and 0.95 for Brahman and B(5)H(3) heifers, respectively) with the photoperiod curve, suggesting the probable influence of this environmental factor on the regulation of estrous cycle activity in heifers with a higher proportion of Bos indicus genes. Thus, the genetic factor needs be taken into account when establishing programs in reproductive management.

Journal Article↗

Evaluation of thresholds for the detection of binding sites for regulatory proteins in Escherichia coli K12 DNA.

BACKGROUND: Sites in DNA that bind regulatory proteins can be detected computationally in various ways. Pattern discovery methods analyze collections of genes suspected to be co-regulated on the evidence, for example, of clustering of transcriptome data. Pattern searching methods use sequences with known binding sites to find other genes regulated by a given protein. Such computational methods are important strategies in the discovery and elaboration of regulatory networks and can provide the experimental biologist with a precise prediction of a binding site or identify a gene as a member of a set of co-regulated genes (a regulon). As more variations on such methods are published, however, thorough evaluation is necessary, as performance may differ depending on the conditions of use. Detailed evaluation also helps to improve and understand the behavior of the different methods and computational strategies. RESULTS: We used a collection of 86 regulons from Escherichia coli as datasets to evaluate two methods for pattern discovery and pattern searching: dyad analysis/dyad sweeping using the program Dyad-analysis, and multiple alignment using the programs Consensus/Patser. Clearly defined statistical parameters are used to evaluate the two methods in different situations. We placed particular emphasis on minimizing the rate of false positives. CONCLUSIONS: As a general rule, sensors obtained from experimentally reported binding sites in DNA frequently locate true sites as the highest-scoring sequences within a given upstream region, especially using Consensus/Patser. Pattern discovery is still an unsolved problem, although in the cases where Dyad-analysis finds significant dyads (around 50%), these frequently correspond to true binding sites. With more robust methods, regulatory predictions could help identify the function of unknown genes.

Bacterial Proteins↗

Identification of simple sequence repeat (SSR) markers for acid detergent fiber in rice straw by bulked segregant analysis.

Rice straw is a significant energy source for ruminant animals. The acid detergent fiber (ADF) content of rice straw is negatively related to intake potential of forages. Therefore, improving the digestibility of rice straw by reducing ADF content is a necessary goal in breeding programs. In the present study, simple sequence repeat (SSR) markers and the bulked segregant analysis (BSA) approach were used to identify molecular markers associated with ADF. A total of 121 BC1F1 plants derived from the cross of JX974 (a cultivar with high ADF, 36.6%) and Dongxiang wild rice (a wild rice with low ADF, 31.3%), with JX974 as a recurrent parent, were used to conduct BSA. Phenotypic analysis showed that ADF displayed a normal distribution in BC1F1 population, indicating the involvement of polygenes. A SSR marker, RM566 on chromosome 9, was identified for ADF. A small linkage map consisting of five markers was constructed by adding four other markers, and a quantitative trait locus (QTL) controlling ADF was mapped at the RM321-RM566 interval, with a distance of 3.9 cM to RM566. This QTL explained 12% of the total phenotypic variation of ADF, and its additive effect was 3%. This study is the first step to map QTL for ADF, one of the plant cell wall components in rice.

Chromosome Mapping↗

Identification of env CRF-10 among HIV variants circulating in rural western Kenya.

As part of a program to determine the genetic diversity of human immunodeficiency virus in rural Kenya, we carried out a molecular analysis of the C2-V3 region of HIV-infected blood samples obtained from 30 antenatal clinic attendees of seven health centers in western Kenya. Direct sequencing was carried out on the envelope C2-V3 region of proviral DNA. On phylogenetic analysis with reference strains, 20 were subtype Al, 2 were subtype D, 1 was subtype C, 1 was subtype G, 1 was CRF-10, 2A/D, 2A/C, and 2 were unclassified. The presence of CRF-10 and the great variety of subtypes and recombinants in such a limited sample size suggest that western Kenya may be a potential hotspot for HIV recombination in the country.

Adolescent↗

Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin.

Human and simian immunodeficiency viruses (HIV and SIV), influenza virus, and hepatitis C virus (HCV) have heavily glycosylated, highly variable surface proteins. Here we explore N-linked glycosylation site (sequon) variation at the population level in these viruses, using a new Web-based program developed to facilitate the sequon tracking and to define patterns (www.hiv.lanl.gov). This tool allowed rapid visualization of the two distinctive patterns of sequon variation found in HIV-1, HIV-2, and SIV CPZ. The first pattern (fixed) describes readily aligned sites that are either simply present or absent. These sites tend to be occupied by high-mannose glycans. The second pattern (shifting) refers to sites embedded in regions of extreme local length variation and is characterized by shifts in terms of the relative position and local density of sequons; these sites tend to be populated by complex carbohydrates. HIV, with its extreme variation in number and precise location of sequons, does not have a net increase in the number of sites over time at the population level. Primate lentiviral lineages have host species-dependent levels of sequon shifting, with HIV-1 in humans the most extreme. HCV E1 and E2 proteins, despite evolving extremely rapidly through point mutation, show limited sequon variation, although two shifting sites were identified. Human influenza A hemagglutinin H3 HA1 is accumulating sequons over time, but this trend is not evident in any other avian or human influenza A serotypes.

Animals↗

Long-range comparison of human and mouse SCL loci: localized regions of sensitivity to restriction endonucleases correspond precisely with peaks of conserved noncoding sequences.

Long-range comparative sequence analysis provides a powerful strategy for identifying conserved regulatory elements. The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis, and it displays a highly conserved expression pattern. We present here a detailed sequence comparison of 193 kb of the human SCL locus to 234 kb of the mouse SCL locus. Four new genes have been identified together with an ancient mitochondrial insertion in the human locus. The SCL gene is flanked upstream by the SIL gene and downstream by the MAP17 gene in both species, but the gene order is not collinear downstream from MAP17. To facilitate rapid identification of candidate regulatory elements, we have developed a new sequence analysis tool (SynPlot) that automates the graphical display of large-scale sequence alignments. Unlike existing programs, SynPlot can display the locus features of more than one sequence, thereby indicating the position of homology peaks relative to the structure of all sequences in the alignment. In addition, high-resolution analysis of the chromatin structure of the mouse SCL gene permitted the accurate positioning of localized zones accessible to restriction endonucleases. Zones known to be associated with functional regulatory regions were found to correspond precisely with peaks of human/mouse homology, thus demonstrating that long-range human/mouse sequence comparisons allow accurate prediction of the extent of accessible DNA associated with active regulatory regions.

Animals↗

Variation in the lamin A/C gene: associations with metabolic syndrome.

OBJECTIVE: Metabolic syndrome is associated with increased risk for cardiovascular disease and type 2 diabetes mellitus (T2DM). The lamin A/C (LMNA) gene, mutations of which cause rare syndromes of severe insulin resistance and dyslipidemia, is located on chromosome 1q21-q24, a region linked to T2DM in several genome wide scans, including in the Old Order Amish. To determine whether polymorphisms in LMNA influence susceptibility to metabolic syndrome and its constituent components. METHODS AND RESULTS: We performed DNA sequence analysis of LMNA. Six single-nucleotide polymorphisms (SNPs) were identified: c.141889C>T (intron 3), c.141906G>T (intron 3), A287A (c.141253T>C; exon 5), c.140353G>A (intron 6), c.139418C>T (intron 8), and H566H (c. 138747C>T; exon 10). In 971 participants from the Amish Family Diabetes Study, the H566H polymorphism of LMNA was associated with metabolic syndrome diagnosed according to National Cholesterol Education Program ATP III criteria and also higher mean fasting triglyceride and lower mean high-density lipoprotein-cholesterol concentrations. However, no differences in allele frequencies were observed for any SNP among participants with T2DM or impaired glucose homeostasis (IGH) and normoglycemic controls. Haplotype analysis showed that >87% of individuals carried 1 of 2 common LMNA haplotypes. There were no significant differences in haplotype frequencies among subjects with metabolic syndrome T2DM, IGH, and controls. CONCLUSIONS: Sequence variation in LMNA may confer modest susceptibility for development of metabolic syndrome and dyslipidemia in the Amish. To determine whether polymorphisms in LMNA influence susceptibility to metabolic syndrome and its constituent components, we performed DNA analysis of polymorphisms in LMNA. The H566H polymorphism was associated with metabolic syndrome and also higher mean fasting triglyceride and lower mean HDL-cholesterol concentrations in the Old Order Amish.

Adult↗

Ethnicity and breast cancer in Hawaii: increased survival but continued disparity.

OBJECTIVES: To examine ethnic variation in survival among 7722 women diagnosed with invasive breast cancer in Hawaii between 1990 and 2002 and to extend previous multivariate analyses by adding a new prognostic variable: estrogen receptor/progesterone receptor (ER/PR) status. DESIGN: Cox regression analysis of retrospective data. SETTING: Population-based data from the Hawaii Tumor Registry, which is part of the National Cancer Institute's Surveillance, Epidemiology, and End Results Program. PARTICIPANTS: 7722 women in 5 ethnic groups--Caucasian, Chinese, Japanese, Filipino, and Native Hawaiian--diagnosed with invasive breast cancer between 1990 and 2002. MAIN OUTCOME MEASURE: Survival, examining death from breast cancer and death from a cause other than breast cancer. RESULTS: Compared to Caucasians, significantly smaller proportions of Japanese and Chinese women and larger proportions of Native Hawaiian and Filipino women were diagnosed in later stages of disease and at earlier ages. The four minority ethnic groups had higher rates of ER+PR+ tumors than Caucasians. For both causes of death, ethnic disparities in survival were reduced, but still existed, after controlling for age, stage, and ER/PR status. Japanese had the highest rates of survival for either cause of death. Native Hawaiians and Filipinos had the lowest rates of survival for breast cancer, and Native Hawaiians and Caucasians had the lowest rates of survival for other causes of death. CONCLUSIONS: Future studies should examine other reasons for continued ethnic differences in breast cancer survival in Hawaii, including socioeconomic status, access to insurance, adequacy of recommended screening frequency, comorbid conditions, treatment appropriateness and compliance, and genetic markers of tumor aggressiveness.

Adult↗

Concurrent validity of the OPCRIT diagnostic system. Comparison of OPCRIT diagnoses with consensus best-estimate lifetime diagnoses.

BACKGROUND: OPCRIT (a suite of computer programs that allow data entry and generate diagnoses according to 12 operational diagnostic systems) is used in a wide range of psychiatric research including both European Science Foundation and NIMH research initiatives in the molecular genetics of mental disorders. We examined its concurrent validity in 100 subjects collected for linkage studies of Bipolar Disorder and Schizophrenia. METHOD: We compared diagnoses generated by OPCRIT from data rated by two trained clinicians with diagnoses made according to consensus, best-estimate, lifetime procedures by the same two raters according to DSM-III-R and RDC classifications. RESULTS: Good to excellent agreement was achieved between OPCRIT diagnoses and those made by consensus best-estimate procedures. CONCLUSIONS: OPCRIT provides a convenient, reliable, rapid and valid approach to polydiagnostic assessment that can be used as an adjunct to conventional (but time consulting) best-estimate consensus diagnostic procedures.

Bipolar Disorder↗

CYP2A6 polymorphisms are associated with nicotine dependence and influence withdrawal symptoms in smoking cessation.

CYP2A6 is the main enzyme that catalyzes nicotine into cotinine. Interindividual differences in nicotine metabolism result at least partially from polymorphic variation of CYP2A6 gene. In this study, we evaluated the influence of CYP2A6 polymorphisms on clinical phenotypes of smoking, such as smoking habit and withdrawal symptoms. Japanese smokers (n = 107) were genotyped for CYP2A6*1, *4 and *9. Consistent with the previous reports, CYP2A6 genotypes have a tendency to correlate with the number of cigarettes per day and with daily intake of nicotine. Interestingly, CYP2A6 high-activity group (CYP2A6*1/*1, *1/*9, *1/*4, *9/*9) smoked the first cigarette of the day earlier than low-activity group (CYP2A6*4/*9, *4/*4), indicating more remarkable nicotine dependence. Furthermore, nicotine withdrawal symptoms were more serious in smoking cessation in CYP2A6 high-activity group. Collectively, CYP2A6 genotypes are related with nicotine dependence, influencing smoking habits and withdrawal symptoms in quitting smoking. It is proposed that individualized smoking cessation program could be designed based on CYP2A6 genotypes.

Aryl Hydrocarbon Hydroxylases↗

Developmental genetics and evolution of symbiotic structures in nitrogen-fixing nodules and arbuscular mycorrhiza.

Genetic and molecular mechanisms of development are compared for two major plant-microbe endosymbioses: N(2)-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi). Development from the primordia formed de novo in root tissues is common for all known types of N(2)-fixing nodules. However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripherical in legumes or central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones). In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia and located intercellularly (infection threads) and intracellularly (symbiosomes). Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps comprising four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation). The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution. The origin of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g., inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules). An analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N(2)-fixing symbioses.

Actinomycetales↗