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No description available.

Genes expressed in developing pea pods were isolated as cDNAs by differential screening techniques. The cDNAs were characterised by DNA sequencing and expression studies were used to investigate the role of isolated cDNAs in pod development. A clone isolated from a pea {Piswn sativum L.) pod cDNA library was shown to contain the complete coding sequence of a polypeptide with considerable homology to various members of the Rab subfamily of small ras-related GTP- binding proteins. Conserved sequences in the isolated clone include the GTP-buiding site, GDP/GTP hydrolysis domain and C-terminal Cys residues involved in membrane attachment. The high percentage amino acid identity suggests that this cDNA may be the product of a gene, designated Psa-rai?, which is the plant counterpart of Rab7. Rab/Ypt proteins are thought to be involved in intracellular transport from the endoplasmic reticulum to the Golgi apparatus and in vesicular transport. If Psa-ra6 is a functional counterpart of yeast YPT7 (RabT) it should be able to complement a yeast YPT7 mutant. An attempt was made to demonstrate that this was the case. Northern analysis showed invariant expression of Psa-rab in developing pods 'with different phenotypes, indicating an essential function for Psa-rab in developing pods. Hybridisation of the Psa-rab cDNA to pea genomic DNA showed that this protein is probably encoded by a single gene. Nearly isogenic pea lines were selected to investigate the genetic basis for lignification of the pea {Pisum sativum L.) pod endocarp. The development of the pod endocarp in the normal and mutant pea pod phenotypes was examined by histochemical staining and light microscopy. The effect of plant growth regulators on endocarp development was also investigated. A pea pod cDNA library representing poly (A)+ RNA purified from L59 pea pods (genotype, PV; phenotype, lignified endocarp) was differentially screened with total cDNA probes prepared from total pod RNA from L59 and LI390 (genotype, PV; phenotype, no lignification of endocarp) pods 4-6 days after flowering (DAP). Two clones, designated pLP18 and pLP19, were selected for further characterisation on the basis of hybridisation to the L59 cDNA probe, but not the LI390 cDNA probe. Northern blotting was used to show that pLP18 represented a mRNA of 0.95 kb. The predicted polypeptide from the LP18 cDNA encoded a putative blue type I copper protein. The expression pattern of LP 18 mRNA in pods and tissues of the experimental pea lines was determined using RT-PCR quantitation. Hybridisation of the cDNA to pea genomic DNA showed that this protein is probably encoded by a single gene. Clone pLP19 yielded a 1.02 kb cDNA fragment encoding the C-terminal portion of an Hsp70 homologue belonging to a highly conserved family of proteins found in a number of eukaryotic species. Northern analysis of RNA from lignified and unlignified pods showed the presence of differentially expressed LP19 transcripts of varying lengths, which may represent differently processed transcripts. Southern analysis confirmed the presence of a single hybridised band in genomic digests of L59, L58 arid LI390. Several mRNA transcripts of the LP19 gene were isolated which differ in the length of their- 3' untranslated regions.

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The objective of this project was to explore the role of nutrients, mainly glucose and fatty acids, in the regulation of insulin gene expression via the complex interaction factors and positive or negative <i>cis</i>-acting DNA elements located throughout the promoter region, which is positioned up to 300bp. Insulin is a vital endocrine hormone, for the regulation of growth and metabolism. Synthesis of insulin occurs in pancreatic cells. The regulation of tissue- and temporal-specific insulin gene expression and its activation in response to extracellular inducers are two fundamental processes that attract many molecular biologists. Therefore to achieve the objective three major approaches were undertaken in this project. 1. Characterizing and identifying the transcription factors that bound to the negative regulatory element (NRE) within the region -258 to -280 of the human insulin promoter. This was done by applying a promising and unique method including a proteomic approach. The major findings propose NRE bind multiple positive and negative factors. 2. Studying in detail the effect of glucose on the factors that bound to the - 230 to -280 region which included NRE binding factors and PDX-1 which binds A-boxes, in various pancreatic rodent cell lines and isolated human islet of Langerhans. This study shows that the glucose stimulating response of the NRE in b cell lines and human islets does not require synergy between adjacent sequence elements such as E2 binding USF and A5 binding the PDX-1. Therefore, the NRE binding factors are able to demonstrate convincingly the existence of short-term transcriptional control of the insulin gene regulated by glucose. 3. Studying the expression of the lipogenic transcription factor SREBPs in the islets of Langerhans and their role in controlling the expression of several genes. The present study provided the first demonstration of the role of SREBPs in mediating the effect of nutrient signals, particularly glucose, on the insulin promoter and its mRNA.

The translation of a series of <I>cat </I>mRNAs containing either the <I>HSP26 </I>5'- leader or various artificial 5'-leaders (Vega Laso <I>et al., </I>1993) were analysed during growth. From this study, the relative translational efficiencies of these mRNAs were shown to vary from 2% to 100% during mid-exponential phase as observed previously (Vega Laso <I>et al.,</I>1993). However, upon analysing the translation of the various <I>cat </I>constructs during growth, their relative translational efficiencies did not change significantly as yeast cells approached stationary phase. A new set of <I>lacZ </I>mRNAs carrying different natural 5'-leaders (<I>PGK1, PYK1, RpL3, Rp29, GDH1, HSP26, HSP12 </I>and <I>TH14</I>) were constructed. These <I>lacZ </I>mRNAs were placed under the control of the promoters taken from genes expressed during different phases of growth (<I>PGK1 </I>and <I>HSP26</I>). Even though the various <I>PGK1-lacZ </I>and <I>HSP26-lacZ </I>mRNAs were translated differentially, the ability of these mRNAs to compete for the translational apparatus did not appear to change as cells entered stationary phase. The translation of a variety of natural mRNAs encoding a wide range of functions was then analysed by determining their polysomal distribution at various points during growth. Irrespective of the growth phase, a large proportion of each mRNA was detected in the polysomal fractions, suggesting that they continued to be translated in stationary phase. Overall, the data strongly suggest that, under the conditions tested, an excess translational capacity exists in stationary phase yeast cells. Hence gene expression may be largely regulated by transcription upon entry to stationary phase.

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No description available.