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PubMed1989

[Important progress for the ophthalmologist in basic genetic research].

Ophthalmologists and human geneticists share a long standing interest in hereditary diseases and anomalies of the eye. Many of the primary genetic eye diseases are known, as ophthalmic symptoms are frequently part of a pleiotropic gene effect or the eye is affected secondarily. Progress in human genetics has also improved the understanding of genetic eye diseases. This can be demonstrated in the analysis of the function of color-vision genes and their abnormalities as well as the retinoblastoma gene. A line can be drawn from early formal analysis of pedigrees to cytogenetic mapping and, finally DNA analysis and sequencing of the involved genes. These advances have not only led to theoretical insights but also have practical applications where the determination of risk is concerned or prenatal diagnosis, genetic counselling, preventive measures and guidance. The retinoblastoma gene has become an important model for a tumor suppressor gene and tumorigenesis in general. Its influence on other types of tumors, such as osteosarcoma and breast cancer must be clarified. Sequencing of the gene opens the possibility of reconstructing the primary gene product by "reverse genetics" and of analyzing its mode of action. DNA analysis has been extended to an increasing number of eye diseases. Precise clinical and genetic analysis and diagnosis are of primary importance, however, for progress in this field.

Chromosome Aberrations↗
PubMed1991

Genetic mapping of the chloroquine-resistance locus on Plasmodium falciparum chromosome 7.

The resurgence of malaria in recent decades has been accompanied by the worldwide spread of resistance to chloroquine, a drug once uncontested as the first-line antimalarial agent because of its efficacy and low toxicity. Chloroquine-resistant strains of Plasmodium falciparum counter the drug by expelling it rapidly via an unknown mechanism. In the absence of explicit biochemical knowledge of this efflux mechanism, reverse genetics provides a powerful approach to the molecular basis of chloroquine resistance. Here we report genetic linkage analysis in which 85 restriction fragment length polymorphism markers were used to examine inheritance of the 14 P. falciparum chromosomes in a laboratory cross between a chloroquine-resistant and a chloroquine-sensitive parasite. Inheritance data from 16 independent recombinant progeny show that the rapid efflux, chloroquine-resistant phenotype is governed by a single locus within an approximately 400-kilobase region of chromosome 7. Identification and characterization of genes within this region should lead to an understanding of the chloroquine-resistance mechanism.

Animals↗
PubMed2005

Multiple gene segments control the temperature sensitivity and attenuation phenotypes of ca B/Ann Arbor/1/66.

Cold-adapted (ca) B/Ann Arbor/1/66 is the influenza B virus strain master donor virus for FluMist, a live, attenuated, influenza virus vaccine licensed in 2003 in the United States. Each FluMist vaccine strain contains six gene segments of the master donor virus; these master donor gene segments control the vaccine's replication and attenuation. These gene segments also express characteristic biological traits in model systems. Unlike most virulent wild-type (wt) influenza B viruses, ca B/Ann Arbor/1/66 is temperature sensitive (ts) at 37 degrees C and attenuated (att) in the ferret model. In order to define the minimal genetic components of these phenotypes, the amino acid sequences of the internal genes of ca B/Ann Arbor/1/66 were aligned to those of other influenza B viruses. These analyses revealed eight unique amino acids in three proteins: two in the polymerase subunit PA, two in the M1 matrix protein, and four in the nucleoprotein (NP). Using reverse genetics, these eight wt amino acids were engineered into a plasmid-derived recombinant of ca B/Ann Arbor/1/66, and these changes reverted both the ts and the att phenotypes. A detailed mutational analysis revealed that a combination of two sites in NP (A114 and H410) and one in PA (M431) controlled expression of ts, whereas these same changes plus two additional residues in M1 (Q159 and V183) controlled the att phenotype. Transferring this genetic signature to the divergent wt B/Yamanashi/166/98 strain conferred both the ts and the att phenotypes on the recombinant, demonstrating that this small, complex, genetic signature encoded the essential elements for these traits.

Adaptation, Physiological↗
PubMed2001

Universal primer set for the full-length amplification of all influenza A viruses.

To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A virus, we need reliable, simple methods that not only characterize partial sequences but analyze the entire influenza A genome. We designed primers based on the fact that the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved between all influenza A viruses and unique for each segment. The primers designed for each segment contain influenza virus specific nucleotides at their 3'-end and non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7, and N9 subtypes, the segment specific sequences of the neuraminidase genes are different. Therefore, we optimized the primer design to allow the amplification of those neuraminidase genes as well. The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.

Child↗
PubMed1996

Discovery of genes involved with learning and memory: an experimental synthesis of Hirschian and Benzerian perspectives.

The biological bases of learning and memory are being revealed today with a wide array of molecular approaches, most of which entail the analysis of dysfunction produced by gene disruptions. This perspective derives both from early "genetic dissections" of learning in mutant Drosophila by Seymour Benzer and colleagues and from earlier behavior-genetic analyses of learning and in Diptera by Jerry Hirsh and coworkers. Three quantitative-genetic insights derived from these latter studies serve as guiding principles for the former. First, interacting polygenes underlie complex traits. Consequently, learning/memory defects associated with single-gene mutants can be quantified accurately only in equilibrated, heterogeneous genetic backgrounds. Second, complex behavioral responses will be composed of genetically distinct functional components. Thus, genetic dissection of complex traits into specific biobehavioral properties is likely. Finally, disruptions of genes involved with learning/memory are likely to have pleiotropic effects. As a result, task-relevant sensorimotor responses required for normal learning must be assessed carefully to interpret performance in learning/memory experiments. In addition, more specific conclusions will be obtained from reverse-genetic experiments, in which gene disruptions are restricted in time and/or space.

Animals↗
PubMed2005

Zebrafish as a model vertebrate for investigating chemical toxicity.

Zebrafish (Danio rerio) has been a prominent model vertebrate in a variety of biological disciplines. Substantial information gathered from developmental and genetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. Although still in its infancy, there is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening. Here we present an overview of the rapidly increasing use of zebrafish in toxicology. Advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanisms of toxicity are highlighted.

Animals↗
PubMed2004

Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation.

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.

Bacterial Proteins↗
PubMed2006

The NS1 gene contributes to the virulence of H5N1 avian influenza viruses.

In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.

Amino Acid Substitution↗
PubMed1992

Familial screening for genetic haemochromatosis by means of DNA markers.

Genetic haemochromatosis (HFE) is a frequent and potentially fatal disease. Early phlebotomies may prevent complications. The recessive gene for HFE is unknown but closely linked to the HLA-A locus. No direct test for homozygosity for HFE is currently available, apart from HLA typing within the family of a patient with confirmed HFE. During a reverse genetic approach to identify the gene, we found three anonymous genomic probes (P3, P5, and I.82) derived from previously cloned YACs and physically mapped in the HLA class I region. P3 and P5 probes recognise 3 loci (P3A, P3B, P5) and I.82 one locus about 100 kb from HLA-A. Using five biallelic polymorphisms (I.82/BglII, P3B/EcoRV, P3B/PstI, P5/HindIII, P3A/PstI), we tested 198 HLA typed subjects from the families of 22 haemochromatosis patients. The information from the five polymorphisms was sufficient to identify unequivocally extended restriction haplotypes in all families. The restriction haplotypes cosegregate with the HFE allele and enable identification of genotypically identical sibs in all families studied. The linked DNA markers described in this article avoid the disadvantages of HLA serological typing and can be used in genetic counselling of HFE families.

DNA↗
PubMed2005

Integrating genotypic and expression data in a segregating mouse population to identify 5-lipoxygenase as a susceptibility gene for obesity and bone traits.

Forward genetic approaches to identify genes involved in complex traits such as common human diseases have met with limited success. Fine mapping of linkage regions and validation of positional candidates are time-consuming and not always successful. Here we detail a hybrid procedure to map loci involved in complex traits that leverages the strengths of forward and reverse genetic approaches. By integrating genotypic and expression data in a segregating mouse population, we show how clusters of expression quantitative trait loci linking to regions of the genome accurately reflect the underlying perturbation to the transcriptional network induced by DNA variations in genes that control the complex traits. By matching patterns of gene expression in a segregating population with expression responses induced by single-gene perturbation experiments, we show how genes controlling clusters of expression and clinical quantitative trait loci can be mapped directly. We demonstrate the utility of this approach by identifying 5-lipoxygenase as underlying previously identified quantitative trait loci in an F(2) cross between strains C57BL/6J and DBA/2J and showing that it has pleiotropic effects on body fat, lipid levels and bone density.

Animals↗
PubMed2005

Modulation of gene expression by siRNA in hematopoietic cells.

RNA interference (RNAi) has been established as a powerful tool for identifying gene function in many biological processes and can be used for genome-wide functional genetic screens in mammalian cells. For such purposes, expression cassettes encoding RNAi triggers can be efficiently introduced into the host cell genome utilizing viral vector systems, resulting in long-term silencing of target gene expression. Transient gene silencing can also be induced by exogenous delivery of suitable RNAi triggers to target cells. However, similarly to other reverse genetic tools, there are technical challenges and limitations associated with RNAi, some of which are specific to hematopoietic cells. In this review we discuss the rational design of effective RNAi triggers, different approaches for their efficient delivery, and the value of RNAi both as a potential therapeutic strategy and as a tool for functional genomics and target validation in hematopoietic cells.

Animals↗
PubMed2002

Zebrafish as a model for hearing and deafness.

The zebrafish is an especially attractive model for the study of the development and function of the vertebrate inner ear. It combines rapid and accessible embryogenesis with a host of genetic and genomic tools for systematic gene discovery and analysis. A large collection of mutations affecting development and function of the ear and a related sensory system, the lateral line, have been isolated; several of these have now been cloned, and at least five provide models for human deafness disorders. Disruption of multiple genes, using both forward and reverse genetic approaches, has established key players--both signaling molecules and autonomous factors--responsible for induction and specification of the otic placode. Vestibular and auditory defects have been detected in adult animals, making the zebrafish a useful system in which to tackle the genetic causes of late onset deafness and vestibular disease.

Animals↗
PubMed1996

An extragenic suppressor of prp24-1 defines genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae.

The temperature-sensitive prp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperature-sensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic to prp21-1. This suppressor, prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that of prp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in the prp24-1 strain. Genetic analysis of the suppressor showed that prp21-2 is not a bypass suppressor of prp24-1. The suppression of prp24-1 by prp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2-U6 snRNA interactions.

Alleles↗
PubMed1992

Colocalization of the gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) in the Xq28 region.

The gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) have both been localized in the Xqter region by genetic mapping and functional expression studies, respectively. In this paper genetic evidence that the DIR locus is localized distal to the DXS305 locus and that the functional gene for the V2 receptor is localized between the markers DXS269 and F8 is presented. These further refinements in the localization of both genes strengthen the assumption that both genes are identical and provide a rationale for cloning the gene by reversed genetics strategies.

Animals↗
PubMed1995

Dissection of memory formation: from behavioral pharmacology to molecular genetics.

Behavioral pharmacology has suggested an intricate, multiphasic pathway of memory consolidation. An integrated molecular pharmacological approach in Drosophila has lent support to this theory recently by dissecting consolidated memory into two genetically distinct components: a cycloheximide-insensitive, anesthesia-resistant memory and a cycloheximide-sensitive long-term memory. In addition, experiments using inducible dominant-negative transgenes in Drosophila or gene knockouts in mice demonstrate a role for cAMP-responsive transcription factors in formation of long-term memory. These studies support the application of reverse-genetic strategies, including the use of temporally specific agonists and antagonists, to advance the functional dissection of memory formation.

Animals↗
PubMed2025

Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection.

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium, a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual life cycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection-selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

Cryptosporidium parvum↗
PubMed2005

Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility.

Mutations of the conserved residues of influenza virus neuraminidase (NA) that are associated with NA inhibitor (NAI) resistance decrease the sialidase activity and/or stability of the NA, thus compromising viral fitness. In fact, clinically derived NAI-resistant variants with different NA mutations have shown different transmissibilities in ferrets (M. L. Herlocher, R. Truscon, S. Elias, H. Yen, N. A. Roberts, S. E. Ohmit, and A. S. Monto, J. Infect. Dis. 190:1627-1630, 2004). Molecular characterization of mutant viruses that have a homogeneous genetic background is required to determine the effect of single mutations at conserved NA residues. We generated recombinant viruses containing either the wild-type NA (RG WT virus) or a single amino acid change at NA residue 119 (RG E119V-NA virus) or 292 (RG R292K-NA virus) in the A/Wuhan/359/95 (H3N2) influenza virus background by reverse genetics. Both mutants showed decreased sensitivity to oseltamivir carboxylate, and the RG R292K-NA virus showed cross-resistance to zanamivir. We also observed differences between the two mutants in NA enzymatic activity and thermostability. The R292K mutation caused greater reduction of sialidase activity and thermostability than the E119V mutation. The NA defect caused by the R292K mutation was associated with compromised growth and transmissibility, whereas the growth and transmissibility of the RG E119V-NA virus were comparable to those of RG WT virus. Our results suggest that NAI-resistant influenza virus variants may differ substantially in fitness and transmissibility, depending on different levels of NA functional loss.

Acetamides↗
PubMed1991

Transcriptional and translational regulation of the expression of the l(2)gl tumor suppressor gene of Drosophila melanogaster.

By structural, biochemical and molecular genetic analyses, we have investigated the different mechanisms that control the expression of the lethal(2) giant larvae gene, a tumor suppressor gene of Drosophila melanogaster. Transcription of the l(2)gl gene is controlled by two highly identical promoters that result from the duplication of the 2.8 kb proximal portion of the gene. These two repeats are 96% homologous. Reverse genetic analysis has shown that each promoter can drive gene expression. In addition to the promoters, both repeats express two or three exons according to the pattern of splicing. The most distal exon in the second repeat is required because it contains the ATG initiating codon at the beginning of the open reading frame. The 3' untranslated region appears to contain motifs that specifically destabilize the transcript. Deletion of this region results in the formation of more stable mRNAs. The l(2)gl gene is characterized by an unusual codon usage that may reflect an enhanced translation efficiency by moderating the strength of pairing between codons and anticodons and may therefore increase the expressivity of this gene. Analysis of the spatio-temporal expression of the l(2)gl transcripts and proteins has shown that transcripts and proteins are produced ubiquitously during early embryogenesis, at a time when expression of the gene is required for preventing tumorigenesis. In the second half of embryogenesis, l(2)gl expression becomes restricted to tissues that do not show any phenotypic alteration in mutant animals. The l(2)gl protein exhibits two distinct intracellular localizations. It is preferentially found free in the cytoplasm but can become associated with the inner face of the plasma membrane where it is restricted to domains facing contiguous cells. In particular, the l(2)gl protein is absent from the basal and apical domains of the plasma membrane. The aim of the current research is directed towards understanding the functional relevance of the l(2)gl protein binding to the plasma membrane and its role in the control of cell proliferation and differentiation.

Animals↗