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PubMed2002

Phyllosphere microbiology.

Aerial plant surfaces harbor large numbers of microbes, some of which are deleterious to plants whereas others are benign or beneficial. Commercial formulations of bacteria antagonistic to plant pathogenic microbes and ice nucleation active bacteria have been utilized as an environmentally safe method to manage plant disease and to prevent frost damage. Molecular genetic tools, microscopic examination and whole-cell bacterial biosensors have provided extensive information on these microbes, their complex associations and their habitat. The aerial habitat influenced by plants, termed the phyllosphere, is particularly amenable to studies of microbial ecology and the information gained should lead to more effective means of plant protection.

Bacteria↗

PubMed2025

Plasma inflammatory proteome profiles identify MASLD among children with overweight or obesity.

BACKGROUND & AIMS: Pediatric metabolic dysfunction-associated steatotic liver disease (MASLD) is increasingly prevalent among children with overweight or obesity, yet its early diagnosis remains a major clinical challenge. This study aimed to identify circulating inflammatory proteins associated with MASLD and to develop a proteomic risk score (ProScore) to improve diagnostic accuracy. METHODS: In this cross-sectional study of 161 children (median age 8.5 years) with overweight or obesity, MASLD was assessed by vibration-controlled transient elastography, with 42 cases identified. Plasma concentrations of 92 inflammation-related proteins were quantified using a high-throughput proximity extension assay. The ProScore was compared with eleven conventional anthropometric/metabolic indices (WHtR, METS-IR, SPISE, PNFI, VAI, LAP, TyG, TyG-ALT, TyG-WC, TyG-WHtR, and TyG-BMI) and a genetic risk score (GRS). Six machine learning algorithms were employed and diagnostic performance was assessed using area under the curve (AUC) with fivefold cross-validation. RESULTS: Fifteen proteins were significantly associated with MASLD. A six-protein panel (FGF-21, CDCP1, CD244, OPG, Flt3L, MCP-1) achieved the highest diagnostic accuracy (AUC = 0.84), exceeding that of all conventional indices (AUC = 0.65-0.78; all P < 0.05). ProScore performance remained robust in school-based validation (AUC = 0.83), with no substantial improvement when combined with conventional indices. Diagnostic accuracy was higher in children with lower GRS (AUC = 0.92) than in those with higher GRS (AUC = 0.80; P = 0.003). CONCLUSIONS: A proteomic signature of systemic inflammation provides accurate, non-invasive identification of MASLD in at-risk children, outperforming conventional metabolic and genetic tools, and may have utility in clinical and public health settings.

Humans↗

PubMed2001

New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori.

BACKGROUND: Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. MATERIALS AND METHODS: We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. RESULTS: After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. CONCLUSIONS: The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori.

Animals↗

PubMed2003

Revamp a model-status and prospects of the Dictyostelium genome project.

International efforts are underway that aim at determining the complete genome sequence of the social amoeba Dictyostelium discoideum. As strategy, a whole chromosome shotgun (WCS) approach was chosen and each of the six Dictyostelium chromosomes was assigned to project partners. The project is well advanced, chromosome 2 was recently published, and it is expected that the sequences of chromosomes 1 and 6 and a gene catalogue for the complete genome will be available at the end of this year. The genome sequence, together with powerful molecular genetic tools, will undoubtedly further accelerate Dictyostelium research into a number of fundamental biological processes that are common to a wide range of eukaryotes. Furthermore, it will constitute the basis for genome-wide functional analyses. The integration of results from these studies should ultimately lead to a better understanding of the complex networks that govern cellular behavior and development.

Animals↗

PubMed2006

Aspergillus oryzae strains with a large deletion of the aflatoxin biosynthetic homologous gene cluster differentiated by chromosomal breakage.

Recently we divided Aspergillus oryzae RIB strains into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-1, aflR, norA, avnA, verB, and vbs), and group 2, having three homologues (avnA, verB, and vbs). Here, partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8 kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups' strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage.

Aflatoxins↗

PubMed2006

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids.

Industrial microorganisms have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. Rational strain development by metabolic engineering is crucial to successful processes, and is based on efficient genetic tools and detailed knowledge of metabolic pathways and their regulation. This review summarizes recent advances in metabolic engineering of the industrial model bacteria Escherichia coli and Corynebacterium glutamicum that led to efficient recombinant biocatalysts for the production of acetate, pyruvate, ethanol, d- and l-lactate, succinate, l-lysine and l-serine.

Amino Acids↗

PubMed2000

Genetics of type 2 diabetes: an overview for the millennium.

Abundant evidence supports a genetic predisposition to both type 2 diabetes and the traits that precede diabetes (insulin resistance and insulin secretion). Unusual causes of diabetes have been identified, including autosomal dominant, single gene forms due to mutations of glucokinase, the hepatocyte nuclear factors, and insulin promoter factor 1. Mitochondrial mutations also may cause type 2 diabetes, but together these causes explain only a small fraction of type 2 diabetes. In contrast, up to 10% of type 2 diabetes, at least in Caucasian populations, may be autoimmune. Animal models of type 2 diabetes support multiple genetic loci. To identify the loci in the remaining 85% of cases, investigators have tested candidate genes in known pathways formutations with some success. However, no candidate identified to date appears to act as a major susceptibility locus. More recently investigators have used linkage approaches to find genes for type 2 diabetes and the prediabetic traits of insulin resistance and insulin secretion. A locus has now been mapped and potential causative variants identified on chromosome 2q, and many other studies are in progress. New genetic tools and the anticipated completion of the human genome project will likely result in the discovery of yet new genes and pathways that may offer new targets for intervention. Whether a better understanding of the pathophysiology can lead to earlier prediction and detection or prevention will depend on the magnitude of risk conferred by individual genes and particular populations.

Animals↗

PubMed2004

Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato.

Fruit constitutes a major component of human diets, providing fiber, vitamins, and phytonutrients. Carotenoids are a major class of compounds found in many fruits, providing nutritional benefits as precursors to essential vitamins and as antioxidants. Although recent gene isolation efforts and metabolic engineering have primarily targeted genes involved in carotenoid biosynthesis, factors that regulate flux through the carotenoid pathway remain largely unknown. Characterization of the tomato high-pigment mutations (hp1 and hp2) suggests the manipulation of light signal transduction machinery may be an effective approach toward practical manipulation of plant carotenoids. We demonstrate here that hp1 alleles represent mutations in a tomato UV-DAMAGED DNA-BINDING PROTEIN 1 (DDB1) homolog. We further demonstrate that two tomato light signal transduction genes, LeHY5 and LeCOP1LIKE, are positive and negative regulators of fruit pigmentation, respectively. Down-regulated LeHY5 plants exhibit defects in light responses, including inhibited seedling photomorphogenesis, loss of thylakoid organization, and reduced carotenoid accumulation. In contrast, repression of LeCOP1LIKE expression results in plants with exaggerated photomorphogenesis, dark green leaves, and elevated fruit carotenoid levels. These results suggest genes encoding components of light signal transduction machinery also influence fruit pigmentation and represent genetic tools for manipulation of fruit quality and nutritional value.

Alleles↗

PubMed2024

The HIV-1 Transcriptional Program: From Initiation to Elongation Control.

A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

HIV-1↗

PubMed1997

The human reelin gene: isolation, sequencing, and mapping on chromosome 7.

The mouse reelin gene (Reln) encodes a novel protein that, when mutated, results in the characteristic reeler phenotype. A key component of this phenotype is the extensive disruption of the organization of many brain structures. Reelin is believed to be an extracellular protein that controls neural cell positioning during brain development. The reelin gene is conserved in many vertebrate species, including humans. To study the role of the reelin homolog in human brain development, we have isolated and characterized the human gene (RELN). Like its murine counterpart, RELN is large, encoding an mRNA of approximately 12 kb. Overlapping cDNA clones containing the entire open reading frame were isolated and sequenced, revealing that the predicted mouse and human proteins are similar in size (388 kD) and that the amino acid and nucleotide sequences are 94.2% and 87.2% identical, respectively. Northern hybridization analyses revealed that RELN is expressed in fetal and postnatal brain as well as liver. The expression of RELN in postnatal human brain was high in the cerebellum. RELN was mapped to human chromosome 7q22, based on both fluorescence in situ hybridization studies and localization within a well-positioned yeast artificial chromosome (YAC) contig. The YAC contig also contains a number of gentic markers. Together, these studies provide the sequence information and genetic tools for performing more detailed analyses of RELN in an attempt to define its role in human brain development and possibly in human disease.

Amino Acid Sequence↗

PubMed2002

Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria.

Complete genome sequences are now available for many bacterial species that lack sophisticated genetic tools. We describe the development of a broad-host-range cre-lox system that allows antibiotic marker recycling in a variety of gram-negative bacteria. This system consists of an allelic exchange vector bearing a kanamycin cassette flanked by loxP sites and a tetracycline-resistant IncP plasmid that provides expression of the Cre recombinase. We demonstrate this system by generating unmarked deletions of genes in two different bacteria, Methylobacterium extorquens AM1 and Burkholderia fungorum LB400. This new antibiotic marker recycling system offers the possibility of creating unmarked mutants in a wide variety of gram-negative bacteria. Furthermore, marker recycling allows the generation of strains bearing multiple genetic manipulations in organisms for which few antibiotic markers are currently available.

Alleles↗

PubMed1992

Identification of the gene for an Escherichia coli poly(A) polymerase.

Many bacterial mRNAs, like those of eukaryotes, carry a polyadenylate sequence at their 3' termini, but neither the function of the bacterial poly(A) moieties nor their biosynthesis have been elucidated. To develop a genetic tool to approach the problem of bacterial poly(A) RNA, we have sought to identify the genes responsible for mRNA polyadenylylation. A poly(A) polymerase was purified to homogeneity from extracts of Escherichia coli and subjected to N-terminal sequence analysis. The 25-residue amino acid sequence obtained was used to design primers for the amplification of the corresponding coding region by the PCR from an E. coli DNA template. A 74-base-pair DNA segment was obtained that matched a region in the pcnB locus of E. coli, a gene that had originally been identified as controlling plasmid copy number [J. Lopilato, S. Bortner & J. Beckwith (1986) Mol. Gen. Genet. 205, 285-290] and was subsequently cloned and sequenced [J. Liu & J. S. Parkinson (1989) J. Bacteriol. 171, 1254-1261]. Direct evidence that the pcnB locus encodes poly(A) polymerase was provided by the observation that a bacterial strain transformed with an inducible expression vector carrying pcnB as a translational fusion produced 100-fold elevated levels of poly(A) polymerase upon induction. No increased poly(A) polymerase activity was observed in cells transformed with expression vectors carrying truncated forms of the pcnB gene. The identification of a gene encoding bacterial poly(A) polymerase opens the way for the study of the biosynthesis and function of bacterial polyadenylylated mRNA.

Amino Acid Sequence↗

PubMed2004

Investigating gene function using mouse models.

Mouse models have become central to investigating and understanding mammalian gene function. Since the development of the first transgenic and knockout models, new methods have been envisioned that both simplify and increase the specificity of mouse models. Combinations of genetic tools have also been applied to dissect gene activities and to provide systems that are both spatially and temporally regulated. Furthermore, novel technologies have been developed that can regulate biological information at each level from creating specific modifications in DNA, to inhibiting gene transcription, to regulating protein activity.

Alleles↗

PubMed1995

Use of transgenics, null mutants, and antisense approaches to study ethanol's actions.

Behavioral and biochemical responses mediating ethanol's actions have been difficult to study in humans and animals because of their complex polygenic nature. Recent progress in the creation of new animal models using recombinant DNA technology has provided a set of genetic tools by which the role of specific candidate genes in ethanol's actions can be examined. These techniques include the creation of transgenic and null mutant mice, as well as manipulation of protein synthesis with antisense treatments. These techniques are reviewed, and their potential applications to alcohol research are discussed.

Alcohol Withdrawal Delirium↗

PubMed1997

DNA methylation and plant development.

The development of genetic tools to alter DNA methylation has led to a deeper understanding of the importance of DNA modification in the life strategies of different eukaryotic organisms. This review focuses on recent findings that demonstrate a role for cytosine methylation in the development of higher plants. The effects of altering DNA modification are considered in the context of unique aspects of plant development.

Arabidopsis↗

PubMed2000

Dissecting the behaviour of transgenic mice: is it the mutation, the genetic background, or the environment?

Increasingly sophisticated and precise molecular genetic tools are applied to mice in order to study the cellular mechanisms underlying higher brain functions, including learning and memory. However, several studies have produced unclear or conflicting results. One reason for this is that performance in the behavioural tests used to assess learning and memory is influenced by various non-cognitive phenomena and can thus easily be affected by mutations through mechanisms unrelated to memory function. We conducted principal component analysis on data from 3003 mice tested using a standardized protocol to demonstrate this for the Morris swimming navigation test, one of the most widely used paradigms to assess memory and hippocampal function. In addition, we present a meta-analysis showing that genetic background and environment alone produce sufficient variation to span the range of most, if not all, behavioural variables and can thus easily mask or fake mutation effects if genetic studies are not designed properly. We suggest that the chance of obtaining useful results is maximized if behavioural deficits are differentiated by combining complementary behavioural protocols and by analysing multiple complementary parameters in each of them. Mutation effects must be contrasted statistically against the influences of genetic background and environment. In many situations, this is most efficiently achieved if (i) mutations are backcrossed to and maintained in one or (preferably) two well-characterized, commonly available inbred strains and (ii) if mutant and wild-type littermates are analysed on a hybrid or mixed genetic background, that is in F1 or F2 generations derived from the inbred stocks.

Animals↗

PubMed2000

Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus.

The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2-10 tandems CA/GT repeats that are interrupted by sequences of 1-10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes.

Animals↗

PubMed2005

An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes.

Aneuploidies are common chromosomal defects that result in growth and developmental deficits and high levels of lethality in humans. To gain insight into the biology of aneuploidies, we manipulated mouse embryonic stem cells and generated a trans-species aneuploid mouse line that stably transmits a freely segregating, almost complete human chromosome 21 (Hsa21). This "transchromosomic" mouse line, Tc1, is a model of trisomy 21, which manifests as Down syndrome (DS) in humans, and has phenotypic alterations in behavior, synaptic plasticity, cerebellar neuronal number, heart development, and mandible size that relate to human DS. Transchromosomic mouse lines such as Tc1 may represent useful genetic tools for dissecting other human aneuploidies.

Aneuploidy↗