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PubMed2005

Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes.

We produced XX<-->XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX* germ cells developed as "eggs" within the seminiferous tubules of XX*<-->XY chimeric testes. These cells were indistinguishable from XX* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes. The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.

Animals↗
PubMed2005

The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era.

Cystic fibrosis (CF) is the most common inherited disorder of childhood. The diagnosis of CF has traditionally been based on clinical features with confirmatory evidence by sweat electrolyte analysis. Since 1989 it has been possible to also use gene mutation analysis to aid the diagnosis. Cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has advanced our understanding of CF, in particular the molecular basis of an expanded CF phenotype. However, because there are over 1000 mutations and 200 polymorphisms, many without recognised effects on CFTR, the molecular diagnosis can be troublesome. This has necessitated measurement of CFTR function with renewed interest in the sweat test. This review provides an overview of the clinical features of CF, the diagnosis and complex genetics. We provide a detailed discussion of the structure and function of CFTR and the classification of CFTR mutations. Sweat electrolyte analysis is discussed, from the physiology of sweating to the rigours of a properly performed sweat test and its interpretation. With this information it is possible to understand the relevance of the sweat test in the genomic era.

Journal Article↗
PubMed2005

A test for ectopic exchange catalyzed by Cre recombinase in maize.

A maize line expressing Cre recombinase as well as the recipient line without the transgene were assayed for evidence of ectopic recombination within the maize genome. Such a test is valuable for understanding the action of Cre as well as for its use to recombine two target lox sites present in the chromosomes. Pollen examination and seed set tests of material expressing Cre provided no evidence of ectopic recombination, which would be manifested in the production of translocations or inversions and result in pollen abortion and reduced seed set. Root-tip chromosome karyotype analysis was also performed on material with and without Cre expression. Chromosomal aberrations in Cre+ material were not observed above the background level.

Chromosome Aberrations↗
PubMed2004

Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome.

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

Animals↗
PubMed2026

Tigecycline-resistant Staphylococcus in waiting pens of a pig slaughterhouse: genomic insights into a food safety alert.

BACKGROUND: The waiting pens of slaughterhouses represent a critical control point in the 'farm-to-fork' continuum, yet their role in the emergence and dissemination of antimicrobial resistance remains understudied. This study investigated tigecycline-resistant Staphylococcus (TRS) in these high-risk zones to assess their prevalence, resistance mechanisms, and transmission dynamics. METHODS: 400 samples were collected from the waiting pens of a pig slaughterhouse in Guangzhou, China. Antimicrobial susceptibility testing, whole-genome sequencing, phylogenetic analysis, and molecular cloning were employed to characterize resistance mechanisms and transmission patterns. RESULTS: 78 TRS strains were isolated and classified into three species, including S. borealis, S. ureilyticus, and S. pasteuri. These isolates exhibited multidrug-resistant phenotypes and carried new mutations in rpsJ and tet(M), which were functionally confirmed to reduce tigecycline susceptibility. Phylogenetic evidence demonstrated clonal transmission between pig farms and the slaughterhouse. The tet(M) gene was located within Staphylococcal cassette chromosome mec elements mediated by IS257, while tet(L) was carried by plasmids formed through IS256/IS257-mediated recombination. CONCLUSIONS: Waiting pens serve as crucial reservoirs for the amplification and dissemination of antimicrobial resistance. Our findings underscore the urgent need for enhanced biosecurity measures, improved waste management, and routine molecular surveillance in these high-risk zones to mitigate the spread of resistance along the food production chain.

Animals↗
PubMed1995

The foot-and-mouth disease virus leader proteinase gene is not required for viral replication.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase has only two known functions: (i) autocatalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. Cleavage of p220 appears to be important for picornavirus replication, since rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220. To explore the role of L in FMDV replication, we generated synthetic FMDV genomes lacking the L gene and tested their viability in cells. Genomes were constructed with the N-terminal Gly codon of VP4 positioned directly following either the first (Lab) or second (Lb) Met codon of the L protein. Cells transfected with synthetic RNAs lacking L and initiating with the Lab Met codon failed to produce viable virus, but cells transfected with RNAs that utilized the second AUG to drive translation of the viral polyprotein produced viable viruses. These leader-deleted viruses produced plaques on BHK cells that were slightly smaller than those produced by wild-type (WT) virus, grew to slightly lower titers than WT virus in BHK cells, shut off host protein synthesis more slowly than WT virus, and were slightly attenuated in mice. These studies indicate that the L proteinase is not essential for FMDV replication and show that in the cells and animals tested the L gene has a limited effect on virus replication.

Amino Acid Sequence↗
PubMed2005

Isolation of genes from plant Y chromosomes.

Few plant species are dioecious and only a small fraction of these species are known to have sex chromosomes. Considerable efforts to isolate sex-linked genes from dioecious Silene latifolia (Caryophillaceae) have resulted in the isolation of surprisingly few sex-linked genes, suggesting that the methods used previously were not efficient in plants. This chapter analyzes the methods that have been and can be used for isolation of genes from plant sex chromosomes. The most successful method used for the isolation of Y-linked genes included the screening of a male complementary DNA (cDNA) library with the probe obtained by degenerate oligonucleotide-primed polymerase chain reaction (PCR) of the microdissected Y chromosomes. However, chromosome microdissection requires sophisticated equipment and is difficult to apply to species with cytologically indistinguishable sex chromosomes. Genome and cDNA library subtraction methods were surprisingly unsuccessful, probably because of low divergence between the homologous X- and Y-linked genes in plants. Segregation testing and genomics-based methods are increasingly popular and are the most promising approaches for isolation of multiple genes from plant sex chromosomes.

Base Sequence↗
PubMed2026

The curious case of sporadic nematode susceptibility in "Tifguard" peanut (Arachis hypogaea): seed mixture or genetic instability?

The Runner-type peanut (Arachis hypogaea L.) cultivar "Tifguard" carries an introgressed chromosomal segment on chromosome A09 from A. cardenasii that confers resistance to root-knot nematode (RKN). Despite this, a proportion of "Tifguard" plants show RKN symptoms, which could plausibly be attributed to seed mixture or outcrossing. However, recent work has shown that cultivated peanut exhibits surprisingly frequent large-scale chromosomal instability (1% to 5%); suggesting that resistance loss could arise from spontaneous structural genomic change. To test these possibilities, we grew foundation seed in an RKN-infested field and collected symptomatic and asymptomatic plants. Lineages derived by single-seed descent were genotyped using the Axiom Arachis 48K SNP array v2 and whole-genome sequencing. Symptomatic lineages lacked the A. cardenasii introgression on chromosome A09 and instead carried the complete endogenous A. hypogaea A09 region at the expected dosage. There was no evidence of large-scale homoeologous exchange, deletion, or other genomic instability affecting this chromosome. Most susceptible plants were closely related to resistant "Tifguard" but lacked the A09 introgression, with a smaller proportion assignable to known nematode-susceptible cultivars, implicating seed mixture with a possible contribution from cross-pollination rather than genomic instability. Because resistance depends on a single major-effect segment, rare events have disproportionate phenotypic impact, placing high demands on genetic purity. For important traits conferred by major loci, marker-based testing across seed-increase stages could verify trait retention directly, and is increasingly practical as marker costs decline.

Arachis↗
PubMed1999

[Modern methods in the diagnosis of tuberculosis: PCR--polymerase chain reaction].

The diagnosis of tuberculosis depends on many factors but essentially on the detection of mycobacteria, their identification to the species level, and their susceptibility to major antituberculous drugs. The classic tests (direct examination-Ziehl-Neelsen, conventional culture and antibiotic susceptibility tests) are combined with modern tests (the molecular tests such as genomic amplification /PCR/ direct from clinical samples). By Polymerase Chain Reaction (PCR) over a billion identical bacteria could be multiplied from a single DNA molecule. These modern tests shortened diagnostics duration from several weeks to one or two days. The sensitivity of PCR test was 97% for Z-N positive, 68% for Z-N negative samples while specificity was 99% either for Z-N positive or negative samples.

DNA, Bacterial↗
PubMed2004

Risk assessment prediction from genome sequences: promises and dreams.

The application of bacterial genomics opens new avenues of research on foodborne pathogens. Foodborne pathogens must be able to colonize their hosts and survive transmission from host to host. Different groups of genes are involved in the processes of survival, colonization, and virulence, and such genes are potential targets for risk assessment and intervention strategies. Filtering from genome sequences the genes relevant to these processes is a major challenge, and although many tools are already available for analyses, this type of data mining is just beginning. For the simplest application, gene comparison, it is important to know how gene function, for instance in virulence, is being defined and tested. In other genomic applications, reserachers look for specific properties or characteristics of (virulence) genes to identify novel gene candidates. Each approach has pitfalls, and gene candidates must be tested in the lab to confirm their function. Models for colonization and virulence are available for most although not all pathogens. Models for survival and stress responses are needed to increase the utilization of genomic approaches to risk assessment. Here, I discuss how genome sequences are likely to help in microbial risk assessment of foodborne pathogens and how dreams may become promises.

Animals↗
PubMed1989

A genomic clone of Zfy-1 from a YDOM mouse strain detects post-meiotic gene expression of Zfy in testes.

In order to obtain a genomic clone of Zfy-1 from a Y chromosome of Mus musculus domesticus (YDOM) origin, we cloned size-fractionated SJL/J DNA in EMBL-4 and selected colonies which hybridized to pDP1007, a human zinc finger Y clone. The specificity of the clone in hybridizations to mouse and human DNA and partial sequencing confirmed that the clone (subcloned as pGZfy1D) was of Zfy-1 origin. Studies on the expression during testicular development of mRNAs hybridizing to the clone suggested that the gene is expressed post-meiotically.

Aging↗
PubMed2005

An alien promoter capture as a primary step of the evolution of testes-expressed repeats in the Drosophila melanogaster genome.

Fertility of Drosophila melanogaster males is impaired due to the disruption of the silencing of the X-linked, testis-expressed, repeated Stellate (Ste) genes. Ste silencing is mediated by symmetric transcription of the paralogous Y-linked repeats and exerted by an RNA interference (RNAi) mechanism. Here we present a scenario for the origin of the Ste genes and their suppressors. The primary intermediate of their evolution emerged as a result of the acquisition of a preformed alien, testis-specific promoter. This intermediate is identified as a chimeric gene containing coding region of an autosomal gene for testis-specific protein kinase CK2. The 5' region of the chimera has been acquired from a member of a family of testis-expressed X-linked genes of unknown function. We propose that the evolution and amplification of the novel chimeric gene have led to the overproduction of the regulatory CK2 subunit in testes. The evolution of the Y-linked descendants of the primary intermediate resulted in the RNAi-mediated suppression of excessive expression of the X-linked paralogs. The newly detected "dead family" of cognate repeats on the Y chromosome has contributed to the evolution of Ste and its suppressors via gene conversion. Our results show that RNAi silencing, considered as a defense against viruses and transposable elements, may be involved in the evolution of eukaryotic genomes.

Animals↗
PubMed2005

Molecular studies on suspect very virulent infectious bursal disease virus genomic RNA samples.

Infectious bursal disease (IBD) associated with high mortality was first observed in Europe in the mid-1980s. The viruses identified in those outbreaks were described as being very virulent infectious bursal disease virus (vvIBDV) strains. These viruses have spread to nearly every continent but have not yet been identified in North America, Australia, and New Zealand. There is a real and immediate concern that the very virulent form of IBDV will continue to spread until it is present on every continent. Genomic RNA samples from IBDV strains suspected of being very virulent were submitted to our laboratory for molecular analysis. Nucleotide sequences of the VP2 gene hypervariable sequence region were determined for 18 of these viruses. A comparison with published vvIBDV sequences indicated that all but one sample (Thai 4) had nucleotide and predicted amino acid sequences consistent with vvIBDV strains. Published sequences and the nucleotide sequences of our 17 putative vvIBDV strains were used to identify unique nucleotides in the VP2 gene. Probe pairs for a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay were designed based on these unique sequences and then used to test the 17 genomic samples that were identified by nucleotide sequencing to be consistent with vvIBDV, plus the one Thai 4 sample that was not consistent with vvIBDV. Using melting temperature (Tm) analysis following real-time RT-PCR, two probe pairs (vv232 and vv256) successfully identified the 17 putative vvIBDV strains and distinguished them from the Thai 4 sample. An additional 26 genomic RNA samples submitted as suspect vvIBDV strains were then tested using the vv232 and vv256 probes. Based on the melting point analysis of these two probes, all 26 samples contained nucleotide sequences consistent with vvIBDV strains. The specificity of the vv232 and vv256 probe pairs was evaluated using 19 non-vvIBDV strains. In every case, the probes distinguished the 19 classic and variant (non-vvIBDV) strains from the putative vvIBDV strains. Diagnostic assays that can reliably identify vvIBDV strains are needed for surveillance programs designed to monitor the spread of these viruses.

Base Sequence↗
PubMed1992

Assignment of the human TYRP (brown) locus to chromosome region 9p23 by nonradioactive in situ hybridization.

The TYRP (brown) locus determines pigmentation and coat color in the mouse. The human homolog of the TYRP locus has been recently identified and shown to encode a 75-kDa transmembrane melanosomal glycoprotein called gp75. The gp75 glycoprotein is homologous to tyrosinase, an enzyme involved in the synthesis of melanin, forming a family of tyrosinase-related proteins. A genomic clone of human gp75 was used to map the human TYRP locus to chromosome 9, region 9p23, by nonradioactive fluorescent in situ hybridization. Specificity of hybridization was tested with a genomic fragment of human tyrosinase that mapped to a distinct site on 11q21. The 9p region has been reported to be nonrandomly altered in human melanoma, suggesting a role for the region near the TYRP locus in melanocyte transformation.

Chromosomes, Human, Pair 9↗
PubMedDate not supplied

Genetic relatedness among Ureaplasma urealyticum serotypes (serovars).

Fourteen serotypes (serovars) are currently recognized in the species Ureaplasma urealyticum. DNA-DNA hybridization tests, cleavage of genomic DNA by restriction endonucleases, and polyacrylamide gel electrophoresis of cell proteins indicate that the U. urealyticum serotypes fall into two genotypically distinct, but related, clusters. Southern hybridization tests of mycoplasmal ribosomal RNA gene probes with DNAs of the various serotypes digested by restriction endonucleases showed that the U. urealyticum chromosome carries two sets (operons) of ribosomal RNA genes. However, the hybridization patterns produced by the serotypes showed differences which were most pronounced among serotypes belonging to the two different clusters.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Molecular epidemiology of Bacillus cereus food poisoning.

OBJECTIVES: To investigate the potential use of DNA techniques in epidemiological diagnosis of Bacillus cereus food poisoning. SUBJECTS: Fifty six B. cereus isolates from milk were studied. DESIGN: The 56 B. cereus isolates were characterised into enterotoxin positive (27 isolates) and enterotoxin negative (29 isolates) using reverse passive latex agglutination technique. SETTING: Plasmid and genomic DNA were isolated from all the B. cereus isolates. The plasmid DNA was analysed by gel electrophoresis, while genomic DNA was used for restriction endonuclease and toxin gene analyses. MAIN OUTCOME MEASURES: Plasmid profile analysis, restriction endonuclease analysis of genomic DNA, and test for bceT and hblA genes by polymerase chain reaction and gene probing. RESULTS: Seventy two per cent of the isolates contained one to five plasmids of molecular sizes between 0.1 to 60 mDa. Restriction analysis of genomic DNA gave different restriction patterns among enterotoxin positive and enterotoxin negative isolates. A polymerase chain reaction assay detected bceT gene in 41.1% of the isolates, 16% of which tested positive for enterotoxin with B. cereus enterotoxin reverse passive latex agglutination (BCET-RPLA) kit, while hblA gene was detected in all the enterotoxin positive isolates. BceT and hblA gene probes detected the respective genes in all the isolates that also tested positive for toxin genes by polymerase chain reaction. CONCLUSION: DNA techniques provide an alternative approach to the diagnosis of enterotoxigenic B. cereus.

Animals↗
PubMed2004

Divergence of conserved non-coding sequences: rate estimates and relative rate tests.

In many eukaryotic genomes only a small fraction of the DNA codes for proteins, but the non-protein coding DNA harbors important genetic elements directing the development and the physiology of the organisms, like promoters, enhancers, insulators, and micro-RNA genes. The molecular evolution of these genetic elements is difficult to study because their functional significance is hard to deduce from sequence information alone. Here we propose an approach to the study of the rate of evolution of functional non-coding sequences at a macro-evolutionary scale. We identify functionally important non-coding sequences as Conserved Non-Coding Nucleotide (CNCN) sequences from the comparison of two outgroup species. The CNCN sequences so identified are then compared to their homologous sequences in a pair of ingroup species, and we monitor the degree of modification these sequences suffered in the two ingroup lineages. We propose a method to test for rate differences in the modification of CNCN sequences among the two ingroup lineages, as well as a method to estimate their rate of modification. We apply this method to the full sequences of the HoxA clusters from six gnathostome species: a shark, Heterodontus francisci; a basal ray finned fish, Polypterus senegalus; the amphibian, Xenopus tropicalis; as well as three mammalian species, human, rat and mouse. The results show that the evolutionary rate of CNCN sequences is not distinguishable among the three mammalian lineages, while the Xenopus lineage has a significantly increased rate of evolution. Furthermore the estimates of the rate parameters suggest that in the stem lineage of mammals the rate of CNCN sequence evolution was more than twice the rate observed within the placental amniotes clade, suggesting a high rate of evolution of cis-regulatory elements during the origin of amniotes and mammals. We conclude that the proposed methods can be used for testing hypotheses about the rate and pattern of evolution of putative cis-regulatory elements.

Animals↗