A high-resolution multistrain haplotype analysis of laboratory mouse genome reveals three distinctive genetic variation patterns

Understanding of the structure and the origin of genetic variation patterns in the laboratory inbred mouse provides insight into the utility of the mouse model for studying human complex diseases and strategies for disease gene mapping. In order to address this issue, we have constructed a multistrain, high-resolution haplotype map for the 99-Mb mouse Chromosome 16 using approximately 70,000 single nucleotide polymorphism (SNP) markers derived from whole-genome shotgun sequencing of five laboratory inbred strains. We discovered that large polymorphic blocks (i.e., regions where only two haplotypes, thus one SNP conformation, are found in the five strains), large monomorphic blocks (i.e., regions where the five strains share the same haplotype), and fragmented blocks (i.e., regions of greater complexity not resembling at all the first two categories) span 50%, 18%, and 32% of the chromosome, respectively. The haplotype map has 98% accuracy in predicting mouse genotypes in two other studies. Its predictions are also confirmed by experimental results obtained from resequencing of 40-kb genomic sequences at 21 distinct genomic loci in 13 laboratory inbred strains and 12 wild-derived strains. We demonstrate that historic recombination, intra-subspecies variations and inter-subspecies variations have all contributed to the formation of the three distinctive genetic signatures. The results suggest that the controlled complexity of the laboratory inbred strains may provide a means for uncovering the biological factors that have shaped genetic variation patterns.     

Host Genetic Variations and Sex Differences Potentiate Predisposition,Severity, and Outcomes of Group A Streptococcus-Mediated Necrotizing Soft Tissue Infections

Host genetic variations play an important role in several pathogenic diseases, and we previously provided strong evidence that these genetic variations contribute significantly to differences in susceptibility and clinical outcomes of invasive group A Streptococcus (GAS) patients, including sepsis and necrotizing soft tissue infections (NSTIs). The goal of the present study was to investigate how genetic variations and sex differences among four commonly used mouse strains contribute to variation in severity, manifestations, and outcomes of NSTIs. DBA/2J mice were more susceptible to NSTIs than C57BL/6J, BALB/c, and CD-1 mice, as exhibited by significantly greater bacteremia, excessive dissemination to the spleen, and significantly higher mortality. Differences in the sex of the mice also contributed to differences in disease severity and outcomes: DBA/2J female mice were relatively resistant compared to their male counterparts. However, DBA/2J mice exhibited minimal weight loss and developed smaller lesions than did the aforementioned strains. Moreover, at 48 h after infection, compared with C57BL/6J mice, DBA/2J mice had increased bacteremia, excessive dissemination to the spleen, and excessive concentrations of inflammatory cytokines and chemokines. These results indicate that variations in the host genetic context as well as sex play a dominant role in determining the severity of and susceptibility to GAS NSTIs.     

Molecular and phylogenetic analysis and vaccine strain match of human influenza A(H3N2) viruses isolated in Northern Greece between 2004 and 2008

Influenza A viruses are characterized by a unique genome structure, causing genetic instability, especially to the genes of haemagglutinin and neuraminidase. The objectives of this research was molecular and phylogenetic analysis of influenza A(H3N2) strains that circulated in Northern Greece since 2004, particularly the identification of sequence variations and the comparison of circulating viruses with vaccine strains. Since 2004 in Northern Greece, a total of 216 clinical samples were positive for influenza virus infections, of which 83 (38.4%) were attributed to influenza A(H3N2). Molecular analysis of the HA genes of 23 isolates showed that all circulating strains had variations at antigenic sites. Receptor binding sites were conserved in 2004–2005 and 2005–2006 strains whereas a variation was observed in all 2006–2007 strains (H195Y). Furthermore, alternative amino acids for sialic acid receptor binding sites were observed in most of the 2004–2006 isolates. Some amino acid substitutions were also observed at the neuraminidase sequences, which however had no effect on the antigenicity of the viruses. Phylogenetic analysis of each year's circulating strains revealed a relatively low match with the vaccine strains A/Fujian/411/02 and A/California/7/04 for 2004–2005 and 2005–2006, respectively, whereas most 2006–2007 isolates match with the vaccine strain, A/Wisconsin/67/05. This year, unique variations were observed at antigenic and glycosylation sites of A/Serres/77/07-like stains. Constant surveillance of yearly variations is of great importance, so that vaccine strains can be evaluated.     

Monoclonal antibody assay for detection of double-stranded RNA and application for detection of group A and non-group A rotaviruses.

Fastidious viruses are generally detected in human body fluids by means of immunoassay or nucleic acid hybridization systems. These approaches can be difficult to apply to the detection of viruses which display variations in antigenic or genetic composition. Rotaviruses are examples of viruses which can display such variations. Recently identified antigenic variants, designated as non-group A rotaviruses, cannot be detected by immunoassays or nucleic acid hybridization assays which utilize reagents directed at group A rotavirus strains. The incomplete understanding of the extent of antigenic and genetic variation has inhibited the development of assay systems for all of the non-group A rotaviruses and has limited the study of their role in human disease. While rotaviruses display genetic variation, they all contain a genome which consists of double-stranded RNA. We utilized a monoclonal antibody to devise a sensitive assay for the measurement of double-stranded RNA and applied it to the detection of a wide range of rotaviruses. We found that the assay could detect double-stranded RNA from as few as 10 PFU of standard strains of group A rotaviruses. The assay system was also capable of detecting double-stranded RNA from several strains of group B rotaviruses isolated from calves, rats, and pigs at levels below those at which viral RNA could be visualized by means of polyacrylamide gel electrophoresis. When applied to the detection of double-stranded RNA in serial stools shed by rotavirus-infected children, the assay system was capable of detecting double-stranded RNA in samples in which antigen could not be detected by immunoassay. The specific nature of the double-stranded RNA detected by this assay system could be determined by the elution of the nucleic acids from the monoclonal antibody and the reaction of the RNA with specific nucleotide probes. The measurement of double-stranded RNA offers a potential method for the sensitive detection of a wide range of rotaviruses and other members of the family Reoviridae.     

Taxonomy of genus Hepacivirus. Application of palindromic nucleotide substitutions for the determination of genotypes of human hepatitis C virus species

The palindromic nucleotide substitutions (PNS) in the 5'-untranslated region (UTR) of Pestivirus RNA have been described as a new, simple and practical method for genotyping. Given the genetic relatedness between Pestivirus and hepatitis C virus species, the application of the method was investigated preliminarily on 180 isolates, including reference strains. The keys for hepatitis C virus identification have been determined at the genus, species, genotype and subtype levels. Secondary structure nucleotide substitutions were characteristics to the genus included in a complex stem-loop structure composed of 112-115 nucleotides. Due to the worldwide importance of hepatitis C virus, and the difficulties encountered in the control of the disease, it is, therefore, important to understand the genetic aspects of the virus. The application of the PNS method might represent an additional useful tool for determining the genetic variations among hepatitis C virus strains. The identification of viral types or subtypes based on genetic changes should improve our understanding of hepatitis C virus and might provide markers for biological differences, such as virulence, and improve understanding of the evolution of the virus.     

Diurnal Variations in Lymphocyte Subpopulations in Lymphoid Organs of Rats with Genetic Catalepsy and Wistar Rats

Parameters of the immune status of the thymus and spleen in rats with genetic catalepsy were lower compared to those in Wistar rats. Diurnal variations in cell subpopulations of lymphoid organs were different in animals of these strains. Behavioral characteristics and neuroendocrine state in rats with genetic catalepsy were associated with specific changes in the immune system and neuroimmune interactions.     

我国各血清型轮状病毒非结构蛋白NSP4基因特征的初步分析

目的 对我国血清学调查中发现的四型（G型）轮状病毒NSP4序列进行测定和分析,了解NSP4的基因类型和变异状况。方法 利用银染测离方法对G1、G2、G3、G9型毒株NSP4 cDNA序列进行测定。结果 四个血清型毒株NSP4的核苷酸和氨基酸序列的同源性分别为78.8%,93.5%;83.0%,95.5%。G1、G3、G9型标本多属Wa组;G2型标本多属KUN组。NSP4的变异主要位于VP4结合域内的135－145位氨基酸。结论 Wa组和KUN组是我国主要的两种NSP4基因型。     

Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.     

Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens

Comparison of DNA from virulent strains of bacterial pathogens with DNA from less virulent or avirulent close relatives allows the identification of those genomic regions that are present only in virulent strains. Such regions are often associated with pathogenicity islands (PIs) and their characterisation can lead to a greater understanding of the pathogenesis of infectious diseases. There is now a large database of bacterial genomic sequences that provides useful reference information with which to compare the genomes of strains that exhibit variations in virulence or host preferences. Subtractive hybridisation (SH) and its sister method, suppression subtractive hybridisation (SSH), are techniques designed to identify those regions present in one genome but absent from another. The application of these techniques has led to the identification of PIs, mobile genetic elements and variations in virulence gene expression in a range of bacterial pathogens.     

Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes

A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.     

Genotyping of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> vaccine strains by multilocus VNTR analysis

In the course of this investigation, we described the genotypic variations across 18 variable chromosomal loci of the five known anthrax bacillus vaccine strains from the collection of microbial cultures of the 48th Central Research Institute of the Ministry of Defense of the Russian Federation. The persistence of inheriting VNTR loci and their potential application in gene identification were estimated in closely related B. anthracis strains. The genetic profile of each of the studied vaccine strains has been determined for all 18 polymorphic loci. Sequencing of their amplification products was performed. We have confirmed that the variations in the electrophoretic mobility of the amplicons are due to the presence of repeats with different numbers of copies in their structure.     

Moss fiber distribution in the hippocampus of chimeric mice

Large differences in hippocampal lamination have been found between several inbred strains of mice. These variations are thought to be caused by differences in developmental processes. We produced aggregation chimaeras between the inbred strains BALB/c and C57BL/6 and studied the lamination patterns in these animals. Next to some intermediate patterns, several patterns not occurring in the donor strains were obtained. These patterns differed also from those found in F1 hybrids. Thus, although the genetic information from both parental strains is present in both F1 and chimaeric mice, different processes occur during development.     

Variation in metabolic enzyme activity of persistent Haemophilus influenzae in respiratory tracts of patients with cystic fibrosis.

Haemophilus influenzae organisms were isolated from sputum specimens prospectively collected from 40 patients with cystic fibrosis during 2 years to study variations in the metabolic enzyme activities of persistent H. influenzae strains as determined by biotyping. In total, 97 distinct H. influenzae strains without variations in their major outer membrane protein (MOMP) patterns and 73 MOMP variants derived from 30 of these distinct strains were obtained. Twelve distinct strains and 42 MOMP variant strains were isolated at multiple time points during the study period, indicating the persistence of these strains. Among the 54 persistent H. influenzae strains, 22 (41%) strains with stable MOMP compositions showed random variations in biotypes. In 39 of 103 (38%) H. influenzae strains, biotype changes coincided with MOMP variations. Biotype variations were the result of both the loss and the acquisition of enzyme activities. The results of the study indicate that changes in metabolic enzyme activity occur randomly during the persistence of H. influenzae organisms in cystic fibrosis patients, irrespective of MOMP variations.     

Influenza B viruses isolated in Uruguay during the 2002-2005 seasons: genetic relations and vaccine strain match

Monitoring antigenic and genetic variations of circulating influenza viruses is critical for the selection of annual vaccine strains. In order to gain insight into the molecular evolution of Influenza B viruses (IBV) isolated in Uruguay in 2002 and 2005 outbreaks, antigenic and phylogenetic studies were carried out for the Hemagglutinin (HA) gene. Antigenic relations among Uruguayan and reference strains isolated elsewhere were performed by means of hemagglutination inhibition assays (HAI). Genetic relations of HA genes from Uruguayan as well as 41 IBV strains isolated elsewhere were established by means of the construction of phylogenetic trees. HAI assays showed a distant antigenic relationship among the 2002 Uruguayan isolates and the 2002 vaccine strain B/Sichuan/379/99. Phylogenetic analysis also revealed a distant genetic relationship among Uruguayan and 2002 vaccine strains. All 2005 IBV Uruguayan strains were both antigenically and genetically related to B/Victoria lineage-viruses. The results of these studies revealed that 2002 IBV Uruguayan strains have a distant antigenic and genetic relation with the 2002 IBV vaccine strain used in Uruguay. The high rate of susceptible individuals in the youngest cohort (<25 years) might be related to the fact that the B/Victoria lineage-viruses were not previously circulating in Uruguay.     

Phenotyping of Staphylococcus aureus reveals a new virulent ST398 lineage

Staphylococcus aureus sequence type (ST)398, which is commonly found as a colonization strain in pig farming, is emerging more frequently as the cause of human infections. In this study, we analysed ST398 of porcine and human origin at the genetic, protein and immunogenic levels. Although genetic analysis of the genes encoding the major virulence factors revealed the presence of the same genes in all strains studied, the results demonstrated spa type crossing alterations in adhesion abilities in addition to a strongly enhanced lysis activity directly linked to impaired clearance attributable to polymorphonuclear leukocytes (PMNs). This change in virulence pattern indicates high heterogenicity in the ST398 pool that is not based on a different genetic make-up, but probably on variations in the genetic regulation systems. These modifications, which are tightly connected to pathogenicity, cannot be detected by conventional diagnostic methods.     

Comparison and application of ribosome spacer DNA amplicon polymorphisms and pulsed-field gel electrophoresis for differentiation of methicillin-resistant Staphylococcus aureus strains.

Analysis of sequences in the fragments of the 16S-23S rRNA intergenic spacer region by the ribosome spacer PCR (RS-PCR) can differentiate strains of methicillin-resistant Staphylococcus aureus (MRSA). We compared this technique with pulsed-field gel electrophoresis (PFGE) for typing MRSA strains and its application during an investigation of an outbreak. A total of 180 isolates of MRSA collected from various hospital laboratories within the United Kingdom and elsewhere were typed by PFGE and RS-PCR. PFGE identified 17 different types among the 180 strains examined, and RS-PCR generated 13 different types. PFGE could detect minor genetic variations among the isolates and could identify the variants which were not discriminated by RS-PCR. Four unique strain types detected by PFGE were not detected by RS-PCR. When applied to typing the outbreak-related strains from the vascular surgery unit at the General Infirmary at Leeds, the results of RS-PCR were identical to those of PFGE. Our results have shown that RS-PCR is a rapid, inexpensive technique that is highly reproducible and almost as discriminatory as PFGE for typing MRSA isolates and should be useful in the local investigation of MRSA outbreaks.     

Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans.

The genetic diversity of 47 enterotoxigenic Escherichia coli (ETEC) strains of serotypes O6:H16, O27:H7, O29:H21, O128ac:H12, and O153:H45, previously isolated from diarrheic patients in Brazil over a period of 15 years, was investigated by random amplification of polymorphic DNA (RAPD). Informative band arrays were obtained with three 10-mer primers with G+C contents of 50, 60, and 70%. Based on the combination of the band profiles generated by the three primers 22 RAPD types were detected, and 5 major clonal clusters, each one with at least 80% identical bands, were established. The clonal clusters corresponded to strains having the same serotype which, in most cases, also had the same virulence factors (colonization factors and toxin types) and outer membrane protein and lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. The results suggested a correlation between phenotypic properties and genetic relatedness of ETEC isolates of human origin and indicated that a reduced number of clonally related strains are found in areas of ETEC endemicity in Brazil. Moreover, the RAPD technique revealed intraserotype-specific variations, undetectable by the combination of several phenotypic typing methods, among the ETEC strains analyzed. These results show that RAPD typing represents a useful tool for population genetics as well as for epidemiological studies of ETEC.     

Genomics of pathogenic bacteria

The general principles of structural and functional organization of genomes in pathogenic bacteria are considered. Main data on the specific features of genomes of Chlamydia trachomatis, Rickettsia prowazekii, Treponema pallidum, Helicobacter pylori, Haemophilus influenzae, Neisseria meningitidis, Vibro cholerae and pathogenic strains of Escherichia coli are summarized. Particular attention is paid to the problems of genetic control of pathogenicity, intraspecies variations in bacterial genomes, to the environmental and evolutionary meaning of horizontal gene transfer. Whether methods for genotyping bacterial strains can be used is discussed.     

Ontogenetic structural and material variations in ovine calcanei: A model for interpreting bone adaptation

Experimental models are needed for resolving relative influences of genetic, epigenetic, and nonheritable functionally induced (extragenetic) factors in the emergence of developmental adaptations in limb bones of larger mammals. We examined regional/ontogenetic morphologic variations in sheep calcanei, which exhibit marked heterogeneity in structural and material organization by skeletal maturity. Cross‐sections and lateral radiographs of an ontogenetic series of domesticated sheep calcanei (fetal to adult) were examined for variations in biomechanically important structural (cortical thickness and trabecular architecture) and material (percent ash and predominant collagen fiber orientation) characteristics. Results showed delayed development of variations in cortical thickness and collagen fiber orientation, which correlate with extragenetic factors, including compression/tension strains of habitual bending in respective dorsal/plantar cortices and load‐related thresholds for modeling/remodeling activities. In contrast, the appearance of trabecular arches in utero suggests strong genetic/epigenetic influences. These stark spatial/temporal variations in sheep calcanei provide a compelling model for investigating causal mechanisms that mediate this construction. In view of these findings, it is also suggested that the conventional distinction between genetic and epigenetic factors in limb bone development be expanded into three categories: genetic, epigenetic, and extragenetic factors. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Dissecting the effects of mtDNA variations on complex traits using mouse conplastic strains

Previous reports have demonstrated that the mtDNA of mouse common inbred strains (CIS) originated from a single female ancestor and that mtDNA mutations occurred during CIS establishment. This situation provides a unique opportunity to investigate the impact of individual mtDNA variations on complex traits in mammals. In this study, we compiled the complete mtDNA sequences of 52 mouse CIS. Phylogenetic analysis demonstrated that 50 of the 52 CIS descended from a single female Mus musculus domesticus mouse, and mtDNA mutations have accumulated in 26 of the CIS. We then generated conplastic strains on the C57BL/6J background for 12 mtDNA variants with one to three functional mtDNA mutations. We also generated conplastic strains for mtDNA variants of the four M. musculus subspecies, each of which contains hundreds of mtDNA variations. In total, a panel of conplastic strains was generated for 16 mtDNA variants. Phenotypic analysis of the conplastic strains demonstrated that mtDNA variations affect susceptibility to experimental autoimmune encephalomyelitis and anxiety-related behavior, which confirms that mtDNA variations affect complex traits. Thus, we have developed a unique genetic resource that will facilitate exploration of the biochemical and physiological roles of mitochondria in complex traits.The mammalian mitochondrial genome (mtDNA) is a closed, circular, double-stranded DNA with genes encoding mitochondrial oxidative phosphorylation (OXPHOS) enzyme complexes that are essential for ATP production (Anderson et al. 1981). In humans, mtDNA variations can be rare pathogenic mutations or deletions that cause maternally inherited mtDNA disorders or common mtDNA variants that lead to functional changes and thus predispose individuals for polygenic diseases (Wallace et al. 1988; Taylor and Turnbull 2005). However, unique genetic characteristics of mammalian mtDNA, for example, the lack of recombination and the transmission as one haplotype, hamper efforts to identify precisely the variations responsible for traits and effects and to define the biochemical and physiological consequences of individual variations. The same characteristics complicate the genetic manipulation of mtDNA. Although several transmitochondrial mice have been produced, a mouse with a single mtDNA mutation has not yet been generated (Pinkert and Trounce 2002).An unexpected observation provided a potential solution to the limitations of mtDNA. In 1982, Ferris et al. (1982) demonstrated that most mouse common inbred strains (CIS) had the same mtDNA RFLP pattern, which suggested that mouse CIS were descended from a single female. This conclusion was confirmed by sequencing the mtDNA of CIS (Johnson et al. 2001; Bayona-Bafaluy et al. 2003; Mathews et al. 2005; Goios et al. 2007), which also demonstrated that several mutations had accumulated during the establishment of the strains, with a higher ratio of nonsynonymous/synonymous mutations than in wild mice (Goios et al. 2007). Those mutations are of potential functional importance, as has been demonstrated in recent studies (Johnson et al. 2001; Mathews et al. 2005; Moreno-Loshuertos et al. 2006). These findings suggest that the systematic production of conplastic strains (mitochondrial substitution strains) with CIS mtDNA carrying one or more functional mutations would constitute a genetic resource to investigate the roles of individual mtDNA variations in complex traits.In the present study, we report the generation of a unique public resource of 16 conplastic strains that carry potential functional mtDNA mutations on a C57BL/6J genetic background. In humans, mtDNA variations are associated with many neurological diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis (MS), and bipolar disorder (Shoffner et al. 1993; Kalman and Alder 1998; Kato et al. 2001; van der Walt et al. 2003). Recently, we reported that the mtDNA allele nt13708A increases susceptibility to MS (Yu et al. 2008). To test the usefulness of this genetic resource, we investigated the conplastic strains with regard to nervous system phenotypes, in particular experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, and anxiety-like behavior as an aspect of bipolar disorder. We show that mtDNA variations affect both phenotypes, which suggests that conplastic strains are a useful genetic resource for investigating the role of mtDNA in complex traits.