Amanda Cottage

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

BackgroundThe floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants.ResultsA method for identifying transformed seedlings in as little as 3.25 d has been developed. Arabidopsis T1 seeds obtained after floral dip transformation are plated on 1% agar containing MS medium and kanamycin, phosphinothricin or hygromycin B, as appropriate. After a 2-d stratification period, seeds are subjected to a regime of 4–6 h light, 48 h dark and 24 h light (3.25 d). Kanamycin-resistant and phosphinothricin-resistant seedlings are easily distinguished from non-resistant seedlings by green expanded cotyledons whereas non-resistant seedlings have pale unexpanded cotyledons. Seedlings grown on hygromycin B differ from those grown on kanamycin and phosphinothricin as both resistant and non-resistant seedlings are green. However, hygromycin B-resistant seedlings are easily identified as they have long hypocotyls (0.8–1.0 cm) whereas non-resistant seedlings have short hypocotyls (0.2–0.4 cm).ConclusionThe method presented here is an improvement on current selection methods as it allows quicker identification of transformed seedlings: transformed seedlings are easily discernable from non-transformants in as little as 3.25 d in comparison to the 7–10 d required for selection using current protocols.

The Arabidopsis plastid-signalling mutant gun1 (genomes uncoupled1) shows altered sensitivity to sucrose and abscisic acid and alterations in early seedling development

Developing seedlings of the Arabidopsis gun1 (genomes uncoupled1) mutant, which is defective in retrograde plastid-to-nucleus signalling, show several previously unrecognized mutant phenotypes. gun1 seedlings accumulated less anthocyanin than wild-type seedlings when grown in the presence of 2% (w/v) sucrose, due to lower amounts of transcripts of early anthocyanin biosynthesis genes in gun1. Norflurazon and lincomycin, which induce retrograde signalling, further decreased the anthocyanin content of sucrose-treated seedlings, and altered the temporal pattern of anthocyanin accumulation. Lincomycin treatment altered the spatial pattern of sucrose-induced anthocyanin accumulation, suggesting that plastids provide information for the regulation of anthocyanin biosynthesis in Arabidopsis seedlings. The temporal pattern of accumulation of LHCB1 transcripts differed between wild-type and gun1 seedlings, and gun1 seedlings were more sensitive to sucrose suppression of LHCB1 transcript accumulation than wild-type seedlings. Growth and development of gun1 seedlings was more sensitive to exogenous 2% sucrose than wild-type seedlings and, in the presence of lincomycin, cotyledon expansion was enhanced in gun1 seedlings compared to the wild type. gun1 seedlings were more sensitive than wild-type seedlings to the inhibition of seedling growth and development by abscisic acid. These observations clearly implicate GUN1 and plastid signalling in the regulation of seedling development and anthocyanin biosynthesis, and indicate a complex interplay between sucrose and plastid signalling pathways.

Early genetic events in HPV immortalised keratinocytes

Cancer of the uterine cervix (CaCx) is the second most common cancer in women worldwide. More than 99% of all cervical cancers contain high‐risk human papillomaviruses (HPVs), with type 16 predominating. HPV infection alone is not sufficient for neoplastic progression; the HPV‐infected cell must undergo additional genetic changes. Cytogenetic analysis of CaCx has been limited due to difficulties in obtaining good‐quality banded chromosome preparations. Oncogenic HPVs immortalise primary genital keratinocytes in vitro, and evidence suggests that the molecular genetic and cytogenetic abnormalities observed in HPV immortalised cells reflect the in vivo changes. Therefore, these lines represent suitable models for HPV‐induced carcinogenesis. We have used both spectral karyotyping (SKY) and multiplex‐FISH (M‐FISH) analysis to identify karyotypic changes in HPV‐16 immortalised keratinocyte cell lines and established CaCx lines. SKY and M‐FISH identified chromosomal abnormalities in all cell lines examined, with a translocation of chromosome 10 or i(10q) occurring in 9 of the 12 cell lines investigated. Further studies with chromosome 10 band‐specific probes identified the translocation event as involving 10q with the breakpoint at 10p11.2 in some cell lines or 10q11.2 in others. The pericentric region of chromosome 10 is known to contain duplicated sequences flanking the centromeric satellites. The duplicated sequences contain many zinc finger transcription factor encoding genes and disruption of these in HPV immortalised cell lines may alter the transcription with consequences for both cellular and viral gene expression.

Bioinformatics methods to predict protein structure and function. A practical approach.

Protein structure prediction by using bioinformatics can involve sequence similarity searches, multiple sequence alignments, identification and characterization of domains, secondary structure prediction, solvent accessibility prediction, automatic protein fold recognition, constructing three-dimensional models to atomic detail, and model validation. Not all protein structure prediction projects involve the use of all these techniques. A central part of a typical protein structure prediction is the identification of a suitable structural target from which to extrapolate three-dimensional information for a query sequence. The way in which this is done defines three types of projects. The first involves the use of standard and well-understood techniques. If a structural template remains elusive, a second approach using nontrivial methods is required. If a target fold cannot be reliably identified because inconsistent results have been obtained from nontrivial data analyses, the project falls into the third type of project and will be virtually impossible to complete with any degree of reliability. In this article, a set of protocols to predict protein structure from sequence is presented and distinctions among the three types of project are given. These methods, if used appropriately, can provide valuable indicators of protein structure and function.

GUN1 (GENOMES UNCOUPLED1) Encodes a Pentatricopeptide Repeat (PPR) Protein Involved in Plastid Protein Synthesis-Responsive Retrograde Signaling to the Nucleus

Plastid-to-nucleus signaling coordinates the expression of nuclear and plastid genes required for the assembly of functional chloroplasts. We have isolated new alleles of gun1 (genomes uncoupled1) by screening EMS-mutagenised lines of Arabidopsis thaliana containing the GFP reporter gene under the control of a tobacco RbcS promoter for GFP expression in the presence of norflurazon, a carotenoid biosynthesis inhibitor, or lincomycin, an inhibitor of plastid translation. gun1 mutants were able to express photosynthesis- related nuclear genes in the presence of lincomycin, unlike other gun (gun2-gun5) mutants. Microarray analysis identified CA1 (CARBONIC ANHYDRASE1) as the gene most responsive to lincomycin in wild-type seedlings, and a CA1 promoter::GFP reporter gene was used to map the gun1 mutation to a 224 kb region on chromosome 2. Sequencing of candidate genes identified GUN1 as At2g31400, encoding a pentatricopeptide repeat (PPR) protein of 918 amino acid residues. gun1-100 contained a point mutation creating a stop codon at amino acid 56, whereas gun1-1 contained a point mutation resulting in an Ala259Val change. GUN1-GFP fusion proteins were targeted to chloroplast nucleoids following microprojectile bombardment of Arabidopsis and tobacco leaves.

THREE RECEPTOR GENES FOR PLASMINOGEN RELATED GROWTH FACTORS IN THE GENOME OF THE PUFFER FISH FUGU RUBRIPES

Plasminogen related growth factors (PRGFs) and their receptors play major roles in embryogenesis, tissue regeneration and neoplasia. In order to investigate the complexity and evolution of the PRGF receptor family we have cloned and sequenced three receptors for PRGFs in the teleost fish Fugu rubripes, a model vertebrate with a compact genome. One of the receptor genes isolated encodes the orthologue of mammalian MET, whilst the other two may represent Fugu rubripes orthologues of RON and SEA. This is the first time three PRGF receptors have been identified in a single species.

AP1 genes in Fugu indicate a divergent transcriptional control to that of mammals.

The draft genomic sequence of the Japanese puffer fish, Fugu rubripes, has now been announced. This is the first complete sequence of a teleost fish and the second available vertebrate sequence, the first being that of human. For the first time, whole-genome comparisons between two vertebrates can be undertaken. Early analysis has suggested that there may be surprising differences in gene regulation between human and fish. In mammals, a gene commonly has several functions, and this may not always be the case in fish. Many gene families comprise more members in fish than they do in mammals, possibly because each fish gene has evolved an individual function. Complexities of gene regulation in mammals has hampered studies of all biological processes from cell proliferation to cell death. Determining the activities of the AP1 transcription factor proteins has been non-trivial. The AP1 complex typically comprises two proteins, a Jun (c-Jun, JunB, and JunD) and a Fos (c-Fos, FosB, Fra1, and Fra2). These proteins can form both homodimers and heterodimers among-themselves and can interact with additional proteins; thus, dissecting their individual roles has been difficult. We have determined that Fugu has more Jun and Fos genes than mammals, and if each proves to have a separate function, then addressing the roles of the individual AP1 proteins in Fugu may be simpler than in human.

SAND, a New Protein Family: From Nucleic Acid to Protein Structure and Function Prediction

As a result of genome, EST and cDNA sequencing projects, there are huge numbers of predicted and/or partially characterised protein sequences compared with a relatively small number of proteins with experimentally determined function and structure. Thus, there is a considerable attention focused on the accurate prediction of gene function and structure from sequence by using bioinformatics. In the course of our analysis of genomic sequence from Fugu rubripes, we identified a novel gene, SAND, with significant sequence identity to hypothetical proteins predicted in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, a Drosophila melanogaster gene, and mouse and human cDNAs. Here we identify a further SAND homologue in human and Arabidopsis thaliana by use of standard computational tools. We describe the genomic organisation of SAND in these evolutionarily divergent species and identify sequence homologues from EST database searches confirming the expression of SAND in over 20 different eukaryotes. We confirm the expression of two different SAND paralogues in mammals and determine expression of one SAND in other vertebrates and eukaryotes. Furthermore, we predict structural properties of SAND, and characterise conserved sequence motifs in this protein family.

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signalling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.1 Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.