Biomedical subjects

George W Haughn

Publications and source records attributed to George W Haughn.

6 recordsLinked to original sources

PubMed2006

TILLING is an effective reverse genetics technique for Caenorhabditis elegans.

BACKGROUND: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetic technique based on the use of a mismatch-specific enzyme that identifies mutations in a target gene through heteroduplex analysis. We tested this technique in Caenorhabditis elegans, a model organism in which genomics tools have been well developed, but limitations in reverse genetics have restricted the number of heritable mutations that have been identified. RESULTS: To determine whether TILLING represents an effective reverse genetic strategy for C. elegans we generated an EMS-mutagenised population of approximately 1500 individuals and screened for mutations in 10 genes. A total of 71 mutations were identified by TILLING, providing multiple mutant alleles for every gene tested. Some of the mutations identified are predicted to be silent, either because they are in non-coding DNA or because they affect the third bp of a codon which does not change the amino acid encoded by that codon. However, 59% of the mutations identified are missense alleles resulting in a change in one of the amino acids in the protein product of the gene, and 3% are putative null alleles which are predicted to eliminate gene function. We compared the types of mutation identified by TILLING with those previously reported from forward EMS screens and found that 96% of TILLING mutations were G/C-to-A/T transitions, a rate significantly higher than that found in forward genetic screens where transversions and deletions were also observed. The mutation rate we achieved was 1/293 kb, which is comparable to the mutation rate observed for TILLING in other organisms. CONCLUSION: We conclude that TILLING is an effective and cost-efficient reverse genetics tool in C. elegans. It complements other reverse genetic techniques in this organism, can provide an allelic series of mutations for any locus and does not appear to have any bias in terms of gene size or location. For eight of the 10 target genes screened, TILLING has provided the first genetically heritable mutations which can be used to study their functions in vivo.

Animals↗

PubMed2006

Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa.

Abstract Ecotilling was used as a simple nucleotide polymorphism (SNP) discovery tool to examine DNA variation in natural populations of the western black cottonwood, Populus trichocarpa, and was found to be more efficient than sequencing for large-scale studies of genetic variation in this tree. A publicly available, live reference collection of P. trichocarpa from the University of British Columbia Botanical Garden was used in this study to survey variation in nine different genes among individuals from 41 different populations. A large amount of genetic variation was detected, but the level of variation appears to be less than in the related species, Populus tremula, based on reported statistics for that tree. Genes examined varied considerably in their level of variation, from PoptrTB1 which had a single SNP, to PoptrLFY which had more than 23 in the 1000-bp region examined. Overall nucleotide diversity, measured as (Total), was relatively low at 0.00184. Linkage disequilibrium, on the other hand, was higher than reported for some woody plant species, with mean r2 equal to 0.34. This study reveals the potential of Ecotilling as a rapid genotype discovery method to explore and utilize the large pool of genetic variation in tree species.

Canada↗

PubMed2005

UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression.

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear "chimeric" at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.

Arabidopsis↗

PubMed2005

BLADE-ON-PETIOLE-dependent signaling controls leaf and floral patterning in Arabidopsis.

NONEXPRESSOR OF PR GENES1 (NPR1) is a key regulator of the plant defense response known as systemic acquired resistance. Accumulation of the signal molecule salicylic acid (SA) leads to a change in intracellular redox potential, enabling NPR1 to enter the nucleus and interact with TGACG sequence-specific binding protein (TGA) transcription factors, which in turn bind to SA-responsive elements in the promoters of defense genes. Here, we show that two NPR1-like genes, BLADE-ON-PETIOLE1 (BOP1) and BOP2, function redundantly to control growth asymmetry, an important aspect of patterning in leaves and flowers. Phenotypes in the double mutant include leafy petioles, loss of floral organ abscission, and asymmetric flowers subtended by a bract. We demonstrate that BOP2 is localized to both the nucleus and the cytoplasm, but unlike NPR1, it is highly expressed in young floral meristems and in yeast interacts preferentially with the TGA transcription factor encoded by PERIANTHIA (PAN). In support of a biological relevance for this interaction, we show that bop1 bop2 and pan mutants share a pentamerous arrangement of first whorl floral organs, a patterning defect that is retained in bop1 bop2 pan triple mutants. Our data provide evidence that BOP proteins control patterning via direct interactions with TGA transcription factors and demonstrate that a signaling mechanism similar to that formally associated with plant defense is likely used for the control of developmental patterning.

Arabidopsis↗

PubMed2005

TILLING without a plough: a new method with applications for reverse genetics.

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms. Its advantages over other reverse genetic techniques include its applicability to virtually any organism, its facility for high-throughput and its independence of genome size, reproductive system or generation time. TILLING is currently being used for the detection of both induced and natural variation in several plant species.

Agriculture↗

PubMed2003

MUCILAGE-MODIFIED4 encodes a putative pectin biosynthetic enzyme developmentally regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis seed coat.

The Arabidopsis seed coat epidermis undergoes a complex process of differentiation that includes the biosynthesis and secretion of large quantities of pectinaceous mucilage, cytoplasmic rearrangement, and secondary cell wall biosynthesis. Mutations in MUM4 (MUCILAGE-MODIFIED4) lead to a decrease in seed coat mucilage and incomplete cytoplasmic rearrangement. We show that MUM4 encodes a putative NDP-l-rhamnose synthase, an enzyme required for the synthesis of the pectin rhamnogalacturonan I, the major component of Arabidopsis mucilage. This result suggests that the synthesis of monosaccharide substrates is a limiting factor in the biosynthesis of pectinaceous seed coat mucilage. In addition, the reduced cytoplasmic rearrangement observed in the absence of a key enzyme in pectin biosynthesis in mum4 mutants establishes a causal link between mucilage production and cellular morphogenesis. The cellular phenotype seen in mum4 mutants is similar to that of several transcription factors (AP2 [APETALA2], TTG1 [TRANSPARENT TESTA GLABRA1], TTG2 MYB61, and GL2 [GLABRA2]). Expression studies suggest that MUM4 is developmentally regulated in the seed coat by AP2, TTG1, and GL2, whereas TTG2 and MYB61 appear to be regulating mucilage production through alternate pathway(s). Our results provide a framework for the regulation of mucilage production and secretory cell differentiation.

Adhesives↗