Joseph M. Bateman

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker.

Abstract Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas.

Temporal control of differentiation by the insulin receptor/tor pathway in Drosophila.

Multicellular organisms must integrate growth and differentiation precisely to pattern complex tissues. Despite great progress in understanding how different cell fates are induced, it is poorly understood how differentiation decisions are temporally regulated. In a screen for patterning mutants, we isolated alleles of tsc1, a component of the insulin receptor (InR) growth control pathway. We find that loss of tsc1 disrupts patterning due to a loss of temporal control of differentiation. tsc1 controls the timing of differentiation downstream or in parallel to the RAS/MAPK pathway. Examination of InR, PI3K, PTEN, Tor, Rheb, and S6 kinase mutants demonstrates that increased InR signaling leads to precocious differentiation while decreased signaling leads to delays in differentiation. Importantly, cell fates are unchanged, but tissue organization is lost upon loss of developmental timing controls. These data suggest that intricate developmental decisions are coordinated with nutritional status and tissue growth by the InR signaling pathway.

Insulin/IGF signalling in neurogenesis

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The performance of a multiwire proportional chamber positron camera for clinical use

The Rutherford Appleton Laboratory clinical positron camera consists of two opposed multiwire proportional chambers (MWPCS) mounted on a rotating gantry capable of performing routine nuclear medicine studies. The system has operated since the end of 1986 with complete reliability. It has a sensitivity of 37 kcps MBq-1 cm3 per axial cm for a 20 cm diameter uniformly filled cylinder of activity. The best spatial resolution obtainable is about 6 mm, although in practice images are smoothed in order to reduce statistical noise with a corresponding decrease in resolution. Cross-plane rays are utilised during image reconstruction and the resulting three-dimensional images exhibit the same spatial resolution in three orthogonal directions over a large cylindrical field of view 15 cm high by 30 cm in diameter. The maximum data-taking rate is limited to 1.5 to 2 kcps at present due to deadtime in the read-out system. The performance of the system is described with particular reference to the problems of imaging with the large fractions of random and scattered events which are a consequence of using large-area detectors. Images of phantoms and patients are presented and proposed modifications to the camera are described.

Measurement of radiation dose to the thyroid using positron emission tomography.

Measurements of the functioning volume of thyroid tissue have been made in 22 patients undergoing radioiodine therapy for thyrotoxicosis, using a prototype multiwire proportional counter positron camera. Tomographic images were produced of the distribution of 124I in the thyroid. Functioning volumes were found to be in the range 21-79 cm3 with volume errors of the order of +/- 4% to +/- 14%. Radioiodine uptake varied from 28% to 98%. Using a value of 6 days for the effective half-life of radioiodine in hyperactive thyroids, radiation doses from a standard therapy administration of 75 MBq of 131I varied from 11 to 48 Gy (compared with a recommended 50-70 Gy). In five cases PET imaging showed a non-uniform distribution of radioiodine in thyroids thought to have uniform uptake from conventional pinhole scintigraphy.

DNA transfer from chloroplast to nucleus is much rarer in Chlamydomonas than in tobacco

By transforming chloroplasts with an antibiotic-resistance gene under the control of a nuclear-specific promoter, we employed a selection scheme to detect the transfer of DNA from the chloroplast to the nucleus in the green alga Chlamydomonas reinhardtii. Among several billion homoplasmic cells tested, we were unable to detect any stable nuclear integration of chloroplast DNA under normal growth conditions or under stress conditions. This contrasts with results reported for the transfer of DNA from chloroplast to nucleus in higher plants and from mitochondrion to nucleus in Saccharomyces cerevisiae. Furthermore, we were unable to detect chloroplast DNA-derived sequences among nuclear genome data for C. reinhardtii, which also contrasts with the situation in higher plants. Taken together, these findings suggest that there is presently little, if any, movement of DNA from chloroplast to nucleus in C. reinhardtii, which may reflect the ultrastructure of the C. reinhardtii cell.

Regulation of Neurogenesis and Epidermal Growth Factor Receptor Signaling by the Insulin Receptor/Target of Rapamycin Pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. We have previously shown that growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. Here we show that the control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. We also show, for the first time in Drosophila, that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors.

The role of mTOR signalling in neurogenesis, insights from tuberous sclerosis complex

Understanding the development and function of the nervous system is one of the foremost aims of current biomedical research. The nervous system is generated during a relatively short period of intense neurogenesis that is orchestrated by a number of key molecular signalling pathways. Even subtle defects in the activity of these molecules can have serious repercussions resulting in neurological, neurodevelopmental and neurocognitive problems including epilepsy, intellectual disability and autism. Tuberous sclerosis complex (TSC) is a monogenic disease characterised by these problems and by the formation of benign tumours in multiple organs, including the brain. TSC is caused by mutations in the TSC1 or TSC2 gene leading to activation of the mechanistic target of rapamycin (mTOR) signalling pathway. A desire to understand the neurological manifestations of TSC has stimulated research into the role of the mTOR pathway in neurogenesis. In this review we describe TSC neurobiology and how the use of animal model systems has provided insights into the roles of mTOR signalling in neuronal differentiation and migration. Recent progress in this field has identified novel mTOR pathway components regulating neuronal differentiation. The roles of mTOR signalling and aberrant neurogenesis in epilepsy are also discussed. Continuing efforts to understand mTOR neurobiology will help to identify new therapeutic targets for TSC and other neurological diseases.

Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain

Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early foetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis we find that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis we find that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, we have identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and have shown that the molecular circuitry required is lineage specific.

Clinical PET with a large area multiwire proportional chamber PET camera

Abstract A large-area multiwire proportional chamber positron camera (MUP-PET) is under evaluation for clinical nuclear medicine use remote from a medical cyclotron. A preliminary physical evaluation shows that the detector has a sensitivity 5–10 times that of a conventional gamma camera but one tenth that of a multicrystal, multiring PET system. The spatial resolution is 6 mm throughout an imaging volume 40 cm diameter by 20 cm axially. The electronic readout presently limits the data acquisition rates to ∼ 2–3 kcps. The system has potential to acquire data at 50 kcps with a further increase in sensitivity of 5–10 and an improved resolution of 3 mm. The camera is under evaluation for clinical use in oncological studies using radiopharmaceuticals labelled with 68 Ga from an in-house generator plus longer-lived radionuclides ( 18 F, 124 I, 55 Co, 68 Ga) from off-site cyclotrons. In particular quantitative measurements of dosimetry in both chemotherapy and radiotherapy will be an important part of the work.